Method for modifying chromosomes

ABSTRACT

The present invention relates to a method for producing a modified foreign chromosome(s) or a fragment(s) thereof, which comprises the steps of: (a) preparing a microcell comprising a foreign chromosome(s) or a fragment(s) thereof, and transferring said foreign chromosome(s) or a fragment(s) into a cell with high homologous recombination efficiency through its fusion with said microcell; (b) in said cell with high homologous recombination efficiency, inserting a targeting vector by homologous recombination into a desired site of said foreign chromosome(s) or a fragment(s) thereof, and/or a desired site of a chromosome(s) derived from said cell with high homologous recombination efficiency, thereby marking said desired site; and (c) in said cell with high homologous recombination efficiency, causing deletion and/or translocation to occur at the marked site of said foreign chromosome(s) or a fragment(s) thereof.

TECHNICAL FIELD

The present invention relates to a method for modifying a chromosome(s)or fragment(s) thereof. The present invention also relates to chimericnon-human animals, a method for producing the same and a method forusing the same. The present invention allows chimeric non-human animalsto retain a foreign giant DNA fragment(s) of at least 1 Mb and toexpress the gene(s) on such a fragment(s), which was impossibleheretofore. Hence, the following becomes possible by using the method.

Production of animals which retain and express a full length of a geneencoding a biologically active substance, for example, a full length ofhuman antibody gene. The biologically active substance, for example, ahuman-type antibody is useful as a pharmaceutical product.

Analysis of functions of human giant genes (e.g., histocompatibilityantigen, dystrophin, etc.) in animals.

Production of model animals with human dominant hereditary disease and adisease due to chromosomal aberration.

The present invention relates to pluripotent cells in which endogenousgenes are disrupted, use of the same, and a method for producingchimeric non-human animals and use of the animals. If a foreignchromosome or a fragment thereof containing a gene encoding a geneproduct identical with or homologous to the gene product encoded by thedisrupted endogenous gene is transferred into the pluripotent cell ofthe present invention as a recipient cell so that a desired functionalcell or a desired chimeric non-human animal is produced from the cell,the transferred gene can be expressed efficiently withoutdifferentiation of the pluripotent cell into a germ cell. Even if a germcell of the non-human animal is affected or the pluripotent cell cannotbe differentiated into a germ cell by the disruption of the endogenousgene or the introduction of a foreign gene, a functional cell, or achimeric non-human animal, a tissue or a cell of the animal can retainand express a foreign giant DNA fragment in excess of the heretoforeunattainable 1 Mb (a million bases) in conditions of a deficiency in theendogenous gene and a decrease in the production of an endogenous geneproduct by producing the desired functional cell or non-human animalfrom the pluripotent cell.

BACKGROUND ART

Techniques of expressing foreign genes in animals, that is, techniquesof producing transgenic animals are used not only for obtaininginformation on the gene's functions in living bodies but also foridentifying DNA sequences that regulate the expression of the genes(e.g., Magram et al., Nature, 315:338, 1985), for developing modelanimals with human diseases (Yamamura et al., “Manual of model mice withdiseases” published by Nakayama Shoten, 1994), for breeding farm animal(e.g., Muller et al., Experientia, 47:923, 1991) and for producinguseful substances with these animals (e.g., Velander et al., P.N.A.S.,89:12003, 1992). Mice have been used the most frequently as hosts forgene transfer. Since mice have been studied in detail as experimentalanimals and the embryo manipulating techniques for mice have beenestablished, they are the most appropriate kind of mammals for genetransfer.

Two methods are known for transferring foreign genes into mice. One isby injecting DNA into a pronucleus of a fertilized egg (Gordon et al.,P.N.A.S., 77:7380, 1980). The other is by transferring DNA into apluripotent embryonic stem cell (hereinafter referred to as “ES cell”)to produce a chimeric mouse (Takahashi et al., Development, 102:259,1988). In the latter method, the transferred gene is retained only in EScell-contributing cells and tissues of chimeric mice whereas it isretained in all cells and tissues of progenies obtained via EScell-derived germ cells. These techniques have been used to produce alarge number of transgenic mice up to now.

However, there had been a limit of the size of DNA capable of beingtransferred and this restricts the application range of thesetechniques. The limit depends on the size of DNA which can be cloned.One of the largest DNA fragments which have ever been transferred is aDNA fragment of about 670 kb cloned into a yeast artificial chromosome(YAC) (Jakobovits et al., Nature, 362:255, 1993). Recently, introductionof YAC containing an about 1 Mb DNA fragment containing about 80 percentof variable regions and portions of constant regions (Cμ, Cδ and Cγ) ofa human antibody heavy-chain was reported (Mendes et al., NatureGenetics, 15:146, 1997). These experiments were carried out by fusing aYAC-retaining yeast cell with a mouse ES cell. Although it is believedthat foreign DNA of up to about 2 Mb can be cloned on YAC (Den Dunnen etal., Hum. Mol. Genet., 1:19, 1992), the recombination between homologousDNA sequences occurs frequently in budding yeast cells and therefore, insome cases, a human DNA fragment containing a large number of repeatedsequences is difficult to retain in a complete form. In fact, certainrecombinations occur in 20-40% of the clones of YAC libraries containinghuman genomic DNA (Green et al., Genomics, 11:584, 1991).

In another method that was attempted, a metaphase chromosome from acultured human cell was dissected under observation with a microscopeand the fragment (presumably having a length of at least 10 Mb) wasinjected into a mouse fertilized egg (Richa et al., Science, 245:175,1989). In the resulting mice, a human specific DNA sequence (Alusequence) was detected but the expression of human gene was notconfirmed. In addition, the procedure used in this method to preparechromosomes causes unavoidable fragmentation of DNA into small fragmentsdue to the use of acetic acid and methanol in fixing the chromosome onslide glass and the possibility that the injected DNA exists as anintact sequence is small.

In any event, no case has been reported to date that demonstratessuccessful transfer and expression in mice of uninterrupted foreign DNAfragments having a length of at least 1 Mb.

Useful and interesting human genes which are desirably transferred intomice, such as genes for antibody (Cook et al., Nature Genetics, 7:162,1994), for T cell receptor (Hood et al., Cold Spring Harbor Symposia onQuantitative Biology, Vol. LVIII, 339, 1993), for histocompatibilityantigen (Carrol et al., P.N.A.S. 84:8535, 1987), for dystrophin (DenDunnen et al., supra) are known to be such that their coding regionshave sizes of at least 1 Mb. Since human-type antibodies are importantas pharmaceutical products, the production of mice which retain andexpress full lengths of genes for human immunoglobulin heavy chains(˜1.5 Mb, Cook et al., supra), and light chain κ (˜3 Mb, Zachau, Gene,135:167, 1993), and light chain λ (˜1.5 Mb, Frippiat et al., Hum. Mol.Genet., 4:983, 1995) is desired but this is impossible to achieve by thestate-of-the-art technology (Nikkei Biotec, Jul. 5, 1993).

Many of the causative genes for human dominant hereditary disease andchromosomal aberration which causes congenital deformity (Down'ssyndrome, etc.) have not been cloned and only the information on theapproximate location of the genes on chromosome is available. Forexample, when a gene of interest is found to be located on a specific Gband, which is made visible by subjecting a metaphase chromosome toGiemsa staining, the G band has usually a size of at least several Mb to10 Mb. In order to transfer these abnormal phenotypes into mice, it isnecessary to transfer chromosomal fragments of at least several Mb thatsurround the causative genes, but this is also impossible with thepresently available techniques.

Hence, it is desired to develop a technique by which a foreign DNAlonger than the heretofore critical 1 Mb can be transferred into a mouseand expressed in it.

DNA longer than 1 Mb can be transferred into cultured animal cells bythe techniques available today. Such transfer is carried outpredominantly by using a chromosome as a mediator. In the case of human,chromosomes have sizes of about 50-300 Mb. Some methods for chromosometransfer into cells have been reported (e.g., McBride et al., P.N.A.S.,70:1258, 1973). Among them, microcell fusion (Koi et al., Jpn. J. CancerRes., 80:413, 1989) is the best method for selective transfer of adesired chromosome. The microcell is a structural body in which one toseveral chromosomes are encapsulated with a nuclear membrane and aplasma membrane. A few chromosomes (in many cases, one chromosome) canbe transferred by inducing a microcell with an agent that inhibits theformation of spindle in a specific kind of cell, separating themicrocell and fusing it with a recipient cell. The resulting librariesof monochromosomal hybrid cells containing only one human chromosomehave been used for mapping known genes and specifying the chromosomes onwhich unknown tumor-suppressor genes and cellular senescence genes exist(e.g., Saxon et al., EMBO J., 5:3461, 1986). In addition, it is possibleto fragment a chromosome by irradiating a microcell with γ-rays and totransfer part of the fragments (Koi et al., Science, 260:361, 1993). Asdescribed above, microcell fusion is considered to be an appropriatemethod for transferring DNA larger than 1 Mb into a cultured animalcell.

The expectation that a mouse could be generated from a cultured cellturned to a real fact when the ES cell which has stable pluripotency wasdiscovered (Evans et al., Nature, 292:154, 1981). Foreign genes, variousmutations and mutations by targeted gene recombination could beintroduced into the ES cell, making it possible to perform a widevariety of genetic modifications in mice (e.g., Mansour et al., Nature,336:348, 1988). The ES cell can be used to produce a mouse having adisrupted target gene by gene targeting techniques. The mouse is matedwith a transgenic mouse having a gene of interest to produce a mousethat expresses the gene of interest efficiently. For example, a mousehaving a disrupted endogenous antibody gene can be mated with a mousehaving a human antibody gene transferred to produce a mouse thatexpresses the human antibody efficiently. A normal diploid cell hasalleles. A transgenic mouse having one allele of an mouse antibodyheavy-chain gene disrupted expresses an increased level of humanantibody in its serum. A mouse having both alleles of mouse antibodyheavy-chain gene disrupted expresses a further remarkably increasedlevel of human antibody (S. D. Wagner et al., Genomics, 35:405-414,1996).

Some researchers have developed a technique in which one allele of atarget gene is disrupted, and then the concentration of a selective drugis increased, thereby deleting both alleles of the target gene (doubleknock-out). However, this technique holds the possibility of a decreasein the ability of the target gene-deficient cell to differentiate into agerm cell because the target gene-deficient cell obtained by thehigh-concentration-selective-culture method is cultured in vivo for along period and because the drug-selection pressure is severe (TakatsuTaki, Experimental Medicine, supplement, Biomanual UP Series BasicTechniques for Immunological Study, Yodo-sha, 1995). In another case, iftwo kinds of selective drugs are used for double knocking-out, forexample, if a neomycin-resistant cell is subjected to a double knock-outtreatment with hygromycin, the double drug-resistant ES cell is rarelydifferentiated to produce a mutant mouse (Watanabe et al., TissueCulture 21, 42-45, 1995). ES cells may lose their differentiation andgrowth capabilities under certain culture conditions. When a genetargeting procedure is performed twice, ES cells do not lose the abilityto differentiate into germ cells of a chimeric mouse but the secondhomologous recombination frequency is extremely low (Katsuki et al.,Experimental Medicine, Vol. 11, No. 20, special number, 1993). Hence,when a target gene-deficient homozygote is produced, particularly whenat least two target genes are targeted, a mouse deficient in each targetgene is produced and then the produced mice are mated with each other toproduce a homozygote mouse deficient in at least two genes (N. Longberget al., Nature, 368:856-859, 1994). If genes to be disrupted exist closeto each other and if a mouse deficient in at least two genes cannot beobtained by mating, heterozygote mice deficient in the two target genesare produced from ES cells and they are mated to produce homodeficientmice (J. H. van Ree et al., Hum Mol Genet. 4:1403-1409, 1995).

An attempt to differentiate a pluripotent ES cell into a functional cellin vitro has been made (T. Nakano et al., Science, 265:1098-1101, 1994,A. J. Potocnik et al., The EMBO Journal, 13:5274-5283, 1994). Thecultivation system used in this attempt, for example, a system in whichthe differentiation into a mature B cell can be induced is expected tobe used in the identification of unknown growth and differentiationfactors which will work in development and differentiation processes ofB cells.

As long as the transfer of giant DNA is concerned, it has been believedthat the size of the aforementioned foreign DNA fragment which can becloned into a YAC vector is the upper limit. The prior art technology ofchromosome transfer for introducing a longer DNA into cultured cells hasnever been applied to gene transfer into mice and this has been believedto be difficult to accomplish (Muramatsu et al., “Transgenic Biology”,published by Kodansha Scientific, p. 143-, 1989).

The reasons are as follows.

The transfer of a human chromosome into a mouse ES cell of a normalkaryotype as a recipient cell would be a kind of transfer of chromosomalaberration. Up to now, it has been believed that genetic aberration atchromosomal levels which is large enough to be recognizable withmicroscopes is generally fatal to the embryogeny in mice (Gropp et al.,J. Exp. Zool., 228:253, 1983 and Shinichi Aizawa, “Biotechnology ManualSeries 8, Gene Targeting”, published by Yodosha, 1995).

Available human chromosomes are usually derived from finitelyproliferative normal fibroblasts or differentiated somatic cells such ascancer cells and the like. It was believed that if a chromosome derivedfrom such a somatic cell was transferred into an undifferentiated EScell, the transferred chromosome might cause differentiation of the EScell or its senescence (Muller et al., Nature, 311:438, 1984; Sugawara,Science, 247:707, 1990).

Only few studies have been reported as to whether a somatic cell-derivedchromosome introduced into an early embryo can function in the processof embryonic development as normally as a germ cell-derived chromosometo ensure the expression of a specific gene in various kinds of tissuesand cells. One of the big differences between the two chromosomes isassumed to concern methylation of the chromosomal DNA. The methylationis changed according to differentiation of cells and its important rolein the expression of tissue-specific genes has been suggested (Ceder,Cell, 53:3, 1988). For example, it has been reported that if amethylated DNA substrate is introduced into a B cell, the methylated DNAis maintained after replication and suppresses a site-directedrecombination reaction which is essential to the activation of anantibody gene (Hsieh et al., EMBO J., 11:315, 1992). In addition, it wasreported that higher levels of de novo methylation occurred inestablished cell lines than in vivo (Antequera et al., Cell, 62:503,1990). On the basis of the studies reported, it could not be easilyexpected that an antibody gene in a human fibroblast or a human-mousehybrid cell which was likely to be methylated at a high level would benormally expressed in a mouse B cell.

It should be noted that there are two related reports of Illmensee etal. (P.N.A.S., 75:1914, 1978; P.N.A.S., 76:879, 1979). One report isabout the production of chimeric mice from fused cells obtained byfusing a human sarcoma cell with a mouse EC cell and the other is aboutthe production of chimeric mice from fused cells obtained by fusing arat liver cancer cell with a mouse EC cell. Many questions about theresults of the experiments in these two reports were pointed out andthus these reports are considered unreliable (Noguchi et al., “MouseTeratoma”, published by Rikogakusha, Section 5, 1987). Although it hasbeen desired to perform a follow-up as early as possible, as of todaywhen 17 years have passed since the publication of these reports,successful reproduction of these experiments has not been reported.Hence, it is believed that foreign chromosomes cannot be retained andthe genes on the chromosomes cannot be expressed in mice by the methoddescribed in these reports.

Under these circumstances, it has been believed to be difficult totransfer a giant DNA such as a chromosomal fragment and express it in ananimal such as mouse. Actually, no study has been made about thisproblem since the Illmensee's reports.

Therefore, an object of the present invention is to provide chimericnon-human animals which retain foreign chromosomes or fragments thereofand express genes on the chromosomes or fragments, and their progenies,and a method for producing the same.

It is also an object of the present invention to provide pluripotentcells containing foreign chromosomes or fragments thereof and a methodfor producing the pluripotent cells.

Another object of the present invention is to provide tissues and cellsderived from the chimeric non-human animals and their progenies.

A further object of the present invention is to provide hybridomasprepared by fusing the cells derived from the chimeric non-human animalsand their progenies with myeloma cells.

A still further object of the present invention is to provide a methodfor producing a biologically active substance that is an expressionproduct of the gene on a foreign chromosome or a fragment thereof byusing the chimeric non-human animals or their progenies, or theirtissues or cells.

It is also an object of the present invention to provide pluripotentcells which can be used as recipient cells into which a foreignchromosome(s) or a fragment(s) thereof is transferred in the productionof chimeric non-human animals retaining the foreign chromosome(s) orfragment(s) thereof and expressing a gene(s) on the foreignchromosome(s) or fragment(s) thereof.

A further object of the present invention is to provide a method forusing the pluripotent cells.

A further object of the present invention is to provide a method formodifying a chromosome(s) or fragment(s) thereof.

DISCLOSURE OF INVENTION

As a result of the various studies conducted to achieve the aboveobjects, the inventors succeeded in transferring chromosomes orfragments thereof derived from human normal fibroblast cells into mouseES cells and obtaining clones which were capable of stable retention ofthe chromosomes or fragments. Moreover, they produced from these ESclones those chimeric mice which retained human chromosomes in normaltissues and which expressed several human genes including human antibodyheavy-chain genes. It has become possible to make that animals retainand express giant DNA fragments by the series of these techniques,although this has been impossible by conventional techniques. Moreover,the inventors succeeded in obtaining embryonic stem cells having both ofantibody heavy-chain and light-chain genes knocked out. The inventorshave also developed a novel method for artificially modifyingchromosomes.

The subject matter of the present invention is as follows:

1. A method for producing a chimeric non-human animal, which comprisespreparing a microcell containing a foreign chromosome(s) or afragment(s) thereof and transferring the foreign chromosome(s) orfragment(s) into a pluripotent cell by fusion with the microcell.2. A method for producing a pluripotent cell containing a foreignchromosome(s) or a fragment(s) thereof, which comprises preparing amicrocell containing a foreign chromosome(s) or a fragment(s) thereofand transferring the foreign chromosomes) or fragment(s) thereof into apluripotent cell by fusion with the microcell.

In the method of item 1 or 2, the foreign chromosome(s) or fragment(s)thereof may be larger than 670 kb, further, at least 1 Mb (one millionbase pairs). The foreign chromosome or fragment thereof may contain aregion encoding an antibody. The microcell containing a foreignchromosome(s) or a fragment(s) thereof may be induced from a hybrid cellprepared by the fusion of a cell from which the foreign chromosome(s) orfragment(s) thereof is(are) derived, with a cell having a high abilityto form a microcell. The microcell containing a foreign chromosome(s) ora fragment(s) thereof may be induced from a cell prepared by a furtherfusion of the microcell induced from the hybrid cell with a cell havinga high ability to form a microcell. The cell from which the foreignchromosome(s) or fragment(s) thereof is(are) derived may be a humannormal diploid cell. The cell having a high ability to form a microcellmay be a mouse A9 cell. The pluripotent cell can be selected fromembryonal carcinoma cells, embryonic stem cells, embryonic germ cellsand mutants thereof. It is preferred that the foreign chromosome orfragment thereof contains a gene of interest and that the pluripotentcell has a disrupted endogenous gene identical with or homologous tosaid gene of interest on the foreign chromosome or fragment thereof. Itis also preferred that the foreign chromosome or fragment thereofcontains at least two genes of interest and that the pluripotent cellhas disrupted endogenous genes identical with or homologous to saidgenes of interest on the foreign chromosome or fragment thereof. In thepluripotent cell, one or both alleles of an endogenous gene identicalwith or homologous to the gene of interest on the foreign chromosome orfragment thereof may be disrupted. The gene of interest may be anantibody gene. The antibody gene may be one or more sets of antibodyheavy-chain and light-chain genes. In the method of item 1 or 2, it ispreferred that the foreign chromosome or fragment thereof contains agene of interest and that the foreign chromosome or fragment thereof istransferred into a pluripotent cell having an endogenous disrupted geneidentical with or homologous to the gene of interest and then, a chimerais produced from the pluripotent cell by using an embryo of a non-humananimal in a strain deficient in an endogenous gene identical with orhomologous to the gene of interest. The non-human animal in a straindeficient in an endogenous gene identical with or homologous to the geneof interest can be produced by homologous recombination in genetargeting. Preferably, the chimeric non-human animal retains the foreignchromosome(s) or fragment(s) thereof, expresses the gene(s) on theforeign chromosome(s) or fragment(s) thereof, and can transmit theforeign chromosome(s) or fragment(s) thereof to its progeny. Thechimeric non-human animal is preferably a mammal, more preferably amouse.

3. A pluripotent cell containing a foreign chromosome(s) or afragment(s) thereof.

In the pluripotent cell, the foreign chromosome(s) or fragment(s)thereof may be larger than 670 kb. In the cell of item 3, the foreignchromosome or fragment thereof may contain a gene of interest and thepluripotent cell has an endogenous disrupted gene identical with orhomologous to the gene of interest on the foreign chromosome or afragment thereof. The foreign chromosome or fragment thereof may containat least two genes of interest and the pluripotent cell has disruptedendogenous genes identical with or homologous to the genes of intereston the foreign chromosome or a fragment thereof. In the pluripotentcell, one or both alleles of an endogenous gene identical with orhomologous to the gene of interest may be disrupted. The foreignchromosome or fragment thereof may contain an antibody gene. Theantibody gene may be one or more sets of antibody heavy-chain andlight-chain genes. The pluripotent cell can be selected from embryonalcarcinoma cells, embryonic stem cells, embryonic germ cells and mutantsthereof. The cells of item 3 can be used to produce chimeric non-humananimals.

4. A chimeric non-human animal retaining a foreign chromosome(s) or afragment(s) thereof and expressing a gene(s) on the foreignchromosome(s) or fragment(s) thereof, or its progeny retaining theforeign chromosome(s) or fragment(s) thereof and expressing the gene(s)on the foreign chromosome(s) or fragment(s) thereof.

In the chimeric non-human animal or its progeny, the foreignchromosome(s) or fragment(s) thereof may be larger than 670 kb. Theforeign chromosome or fragment thereof may contain a gene of interestand the animal may have an endogenous disrupted gene identical with orhomologous to the gene of interest. The foreign chromosome or fragmentthereof may contain at least two genes of interest and the animal mayhave disrupted endogenous genes identical with or homologous to saidgenes of interest. In the chimeric non-human animal or its progeny, oneor both alleles of an endogenous gene identical with or homologous tothe gene of interest may be disrupted. The gene of interest may be anantibody gene. The antibody gene may be one or more sets of antibodyheavy-chain and light-chain genes.

5. A non-human animal which can be produced by mating the chimericnon-human animals or their progenies of item 4, said non-human animalretaining the foreign chromosome(s) or fragment(s) thereof andexpressing the gene(s) on the foreign chromosome(s) or fragment(s)thereof, or its progeny retaining the foreign chromosome(s) orfragment(s) thereof and expressing the gene(s) on the foreignchromosome(s) or fragment(s) thereof.6. A non-human animal retaining the foreign chromosome(s) or fragment(s)thereof and expressing a gene(s) on the foreign chromosome(s) orfragment(s) thereof, which can be produced by mating the chimericnon-human animal or its progeny of item 4, or the non-human animal orits progeny of item 5, with a non-human animal in a strain deficient insaid gene(s) or a gene homologous thereto, or its progeny retaining theforeign chromosome(s) or fragment(s) thereof and expressing the gene(s)on the foreign chromosome(s) or fragment(s) thereof.7. A tissue from the chimeric non-human animal or its progeny of item 4or from the non-human animal or its progeny of item 5 or from thenon-human animal or its progeny of item 6.8. A cell from the chimeric non-human animal or its progeny of item 4 orfrom the non-human animal or its progeny of item 5 or from the non-humananimal or its progeny of item 6.

The cell may be a B cell, a primary culture cell derived from an animaltissue or a cell fused with an established cell.

9. A hybridoma prepared by the fusion of the B cell with a myeloma cell.10. A method for producing a biologically active substance, whichcomprises expressing the gene(s) on the foreign chromosome(s) orfragment(s) thereof in the chimeric non-human animal or its progeny ofitem 4, the non-human animal or its progeny of item 5 or the non-humananimal or its progeny of item 6, or a tissue or a cell thereof, andrecovering the biologically active substance as an expression product.

In the method, the cell of the chimeric non-human animal may be a Bcell. The B cell may be immortalized by fusion with a myeloma cell. Thechimeric non-human animal cell may be fused with a primary culture cellderived from an animal tissue or fused with an established cell line.The biologically active substance may be an antibody. The antibody ispreferably an antibody of a mammal, more preferably a human antibody.

11. A biologically active substance which can be produced by the methodof item 10.12. A non-human animal retaining at least one human antibody gene largerthan 670 kb and expressing the gene.

The non-human animal of item 12 preferably retains at least one humanantibody gene of at least 1 Mb and expresses the gene. The humanantibody gene may be a human heavy-chain gene, a human light-chain κgene, a human light-chain λ gene, or a combination thereof. Thenon-human animal of item 12 may be deficient in a non-human animalantibody gene identical with or homologous to the human antibody gene.The deficiency of non-human animal antibody gene may be caused bydisrupting the non-human animal antibody gene by homologousrecombination.

13. A hybridoma prepared by the fusion of a spleen cell of the non-humananimal of item 12 with a myeloma cell.14. An antibody produced by the hybridoma of item 13.15. A non-human animal expressing at least one class or subclass ofhuman antibody.

The non-human animal of item 15 may be deficient in an endogenousantibody gene identical with or homologous to the expressed humanantibody gene. The class or subclass of human antibody may be IgM, IgG,IgE, IgA, IgD or a subclass, or a combination thereof.

16. A non-human animal retaining a foreign DNA(s) larger than 670 kb andexpressing a gene(s) on the foreign DNA(s).

The non-human animal of item 16 may be deficient in an endogenous geneidentical with or homologous to the expressed gene on the foreign DNA.The non-human animal of item 16 may retain a foreign DNA(s) of at least1 Mb and express the gene(s) on the foreign DNA(s) The non-human animalmay be deficient in an endogenous gene identical with or homologous tothe expressed gene on the foreign DNA.

17. A method for producing a transgenic non-human animal, whichcomprises preparing a microcell containing a foreign chromosome(s) or afragment(s) thereof, transferring the foreign chromosome(s) orfragment(s) into a cultured cell derived from a blastcyst by fusion withthe microcell and transplanting the nucleus of the cultured cell into anenucleated unfertilized egg.18. A pluripotent cell in which at least two endogenous genes aredisrupted.

In the cell of item 18, each of the endogenous genes may be disrupted inone or both alleles. The disrupted endogenous genes may be antibodygenes. The disrupted antibody genes may be antibody heavy-chain andlight-chain genes. The pluripotent cell can be selected from embryonalcarcinoma cells, embryonic stem cells, embryonic germ cells and mutantsthereof.

19. A method of producing the cell of item 18 by at least two homologousrecombinations.

The method of item 19 may comprise the steps of: disrupting one alleleof the endogenous gene in the pluripotent cell by homologousrecombination using a drug-resistant marker gene;

culturing the pluripotent cell in the presence of the drug to selectdrug-resistant cells; andscreening the selected drug-resistant cells to yield a cell in whichboth alleles of the endogenous gene have been disrupted.

In the method of item 19, one allele of the endogenous gene in thepluripotent cell may be disrupted by homologous recombination using adrug-resistant marker gene and the other allele of the endogenous genemay be disrupted by another homologous recombination using adrug-resistant marker gene. The same drug-resistant marker gene may beused in the two homologous recombinations. Alternatively, differentdrug-resistant marker genes may be used in the two homologousrecombinations.

Furthermore, the present invention provides a method of using thepluripotent cell as a recipient cell into which a foreign gene(s) or afragment(s) thereof, or a foreign chromosome(s) or a fragment(s) thereofare to be transferred. The foreign gene(s) or fragment(s) thereof may beincorporated in a vector such as a plasmid, a cosmid, YAC or the like.Alternatively, the foreign chromosome(s) or fragment(s) thereof may becontained in a microcell. The foreign chromosome(s) or fragment(s)thereof is preferably, but not limited to, one that contains a gene(s)identical with or homologous to the endogenous gene(s) disrupted in thepluripotent cell. The term “homologous gene” means herein a geneencoding the same kind of protein or a protein having a similar propertyin the same or different species of a given organism.

Moreover, the present invention provides a method of using thepluripotent cell for producing a chimeric non-human animal.

The present invention also provides a method of producing a pluripotentcell containing a foreign chromosome(s) or a fragment(s) thereof, whichcomprises the steps of:

preparing a microcell containing the foreign chromosome(s) orfragment(s) thereof; andfusing the microcell with said pluripotent cell having at least twoendogenous genes disrupted, whereby said foreign chromosome(s) orfragment(s) thereof is transferred into said pluripotent cell.

The present invention further provides a method of producing a chimericnon-human animal, which comprises the steps of:

preparing a microcell containing a foreign chromosome(s) or afragment(s) thereof; andfusing the microcell with said pluripotent cell having at least twoendogenous genes disrupted, whereby said foreign chromosome(s) orfragment(s) thereof is transferred into said pluripotent cell.

In the aforementioned two methods, the foreign chromosome(s) orfragment(s) thereof may have a length(s) of at least 1 Mb (100 millionbase pairs). The foreign chromosome(s) or a fragment(s) thereof maycontain a region encoding an antibody. The microcell containing theforeign chromosome(s) or fragment(s) thereof may be induced from ahybrid cell prepared by the fusion of a cell containing the foreignchromosome(s) or fragment(s) thereof, with a cell having a high abilityto form a microcell. The microcell containing the foreign chromosome(s)or fragment(s) thereof may be induced from a cell prepared by a furtherfusion of the microcell induced from the hybrid cell, with a cell havinga high ability to form a microcell. The cell containing the foreignchromosome(s) or fragment(s) thereof may be a human normal diploid cell.The cell having a high ability to form a microcell may be a mouse A9cell. In the methods of producing a chimeric non-human animal, a foreignchromosome(s) or a fragment(s) thereof containing gene(s) identical withor homologous to the endogenous gene(s) disrupted in the pluripotentcell may be transferred into the pluripotent cell having the disruptedat least two endogenous genes and then, a chimera of the cell with anembryo of a non-human animal in a strain deficient in a gene(s)identical with or homologous to said endogenous gene(s) may be prepared.The chimeric non-human animal deficient in a gene identical with orhomologous to the endogenous gene disrupted in said pluripotent cell maybe produced by homologous recombination in gene targeting. The chimericnon-human animal may be such that it retains the foreign chromosome(s)or fragment(s) thereof, expresses a gene(s) on the foreign chromosome(s)or fragment(s) thereof, and can transmit the foreign chromosome(s) orfragment(s) thereof to its progeny. The chimeric non-human animal may bea mammal, preferably a mouse.

The present invention also provides a pluripotent cell containing aforeign chromosome(s) or a fragment(s) thereof, which is obtainable by amethod of producing a chimeric non-human animal, which method comprisesthe steps of:

preparing a microcell containing the foreign chromosome(s) orfragment(s) thereof; andfusing the microcell with said pluripotent cell having at least twoendogenous genes disrupted, whereby said foreign chromosome(s) orfragment(s) thereof is transferred into said pluripotent cell. Thepresent invention further provides a method of using the cell forproducing a chimeric non-human animal.

The present invention also provides a chimeric non-human animalretaining a foreign chromosome(s) or a fragment(s) thereof andexpressing the gene(s) on the foreign chromosome(s) or fragment(s)thereof, which is obtainable by one of the aforementioned methods ofproducing a chimeric non-human animal, or its progeny. The presentinvention also provides a non-human animal retaining a foreignchromosome(s) or a fragment(s) thereof and expressing the gene(s) on theforeign chromosome(s) or fragment(s) thereof which is obtainable bymating between the chimeric non-human animals or its progenies, or itsprogeny. The present invention further provides a tissue from theaforementioned chimeric non-human animal or its progeny, or theaforementioned non-human animal or its progeny.

The present invention still more provides a cell from the aforementionedchimeric non-human animal or its progeny, or the aforementionednon-human animal or its progeny. The cell may be a B cell.

The present invention also provides a hybridoma prepared by the fusionof the cell from the aforementioned chimeric non-human animal or itsprogeny, or the aforementioned non-human animal or its progeny with amyeloma cell.

The present invention provides a non-human animal or its progenyretaining a foreign chromosome(s) or a fragment(s) thereof andexpressing a gene(s) on the foreign chromosome(s) or fragment(s)thereof, which is obtainable by mating said chimeric non-human animal orits progeny or said non-human animal or its progeny retaining theforeign chromosome(s) or fragment(s) thereof and expressing the gene(s)on the foreign chromosome(s) or fragment(s) thereof, with a non-humananimal in a stain deficient in a gene(s) identical with or homologous tosaid gene(s).

Furthermore, the present invention provides a method of producing abiologically active substance, which comprises expressing a gene(s) on aforeign chromosome(s) or a fragment in the chimeric non-human animal orits progeny, or the non-human animal or its progeny, or a tissue or acell thereof and recovering the biologically active substance as theexpression product. The cell of the chimeric non-human animal or itsprogeny, or the non-human animal or its progeny may be a B cell. The Bcell may be immortalized by fusion with a myeloma cell. The biologicallyactive substance may be an antibody. The antibody may be an antibody ofmammal, preferably a human antibody.

Moreover, the present invention provides a method of producing abiologically active substance, which comprises expressing a gene(s) on aforeign chromosome(s) or a fragment in a offspring or a tissue and acell thereof, wherein the offspring is produced by mating the chimericnon-human animal or its progeny, or the non-human animal or its progenyretaining the foreign chromosome(s) or fragment(s) thereof with anon-human animal in a strain deficient in a gene identical with orhomologous to said genes, and expressing the gene(s) on the foreignchromosome(s) or fragment(s) thereof, and recovering the biologicallyactive substance as the expression product.

The present invention also provides a vector comprising a foreignchromosome(s) for use in gene transfer into a non-human animal and anon-human animal cell. The foreign chromosome(s) is preferably one fromhuman, more preferably a human chromosome #14 fragment. The non-humananimal is preferably a mouse.

The term “allele” is used herein.

The term “homologous gene” means herein a gene encoding the same kind ofprotein or a protein having a similar property in the same or differentspecies of a given organism.

As used herein, the term “foreign chromosome” means a chromosome whichis exogenously introduced into a target cell. When producing a cellcomprising a modified foreign chromosome(s) or a fragment(s) thereof,the introduced chromosome is a chromosome exogenous to the cell wherechromosome modification occurs (for example, a cell with high homologousrecombination efficiency, such as a chicken DT-40 cell) Further, whenproducing a chimeric non-human animal comprising a modified foreignchromosome(s) or fragment(s) thereof, the introduced chromosome is achromosome exogenous to a pluripotent non-human animal cell (forexample, an ES cell) to which a modified chromosome is transferred, inaddition to a cell where chromosome modification occurs. Alternatively,when producing a non-human animal comprising a modified foreignchromosome(s) or fragment(s) thereof, the introduced chromosome is achromosome exogenous to a cell derived from non human animals (forexample, a culture cell derived from an embryo, a blastocyst, a fetus,or an adult, or a fetal fibrobrast) to which a modified chromosome(s) istransferred, in addition to a cell where chromosome modification occurs.

The present invention allows a gene on the chromosome(s) or fragment(s)thereof to be expressed in a chimeric non-human animal or a non-humananimal that is produced from a pluripotent cell, into which a foreignchromosome(s) or fragment(s) thereof is transferred. Species from whichtarget cells are derived and species from which foreign chromosomes arederived are not specifically limited. Both species of a target cell andof a foreign chromosome be the same or may be different.

Further summary of the present invention is as follows.

-   1. A method for producing a cell comprising a modified foreign    chromosome(s) or a fragment(s) thereof, which comprises the steps    of:-   (a) preparing a microcell comprising a foreign chromosome(s) or a    fragment(s) thereof, and transferring said foreign chromosome(s) or    a fragment(s) thereof into a cell with high homologous recombination    efficiency through its fusion with said microcell;-   (b) in said cell with high homologous recombination efficiency,    inserting a targeting vector by homologous recombination into a    desired site of said foreign chromosome(s) or a fragment(s) thereof    and/or a desired site of a chromosome(s) derived from said cell with    high homologous recombination efficiency, thereby marking the    desired site; and-   (c) in said cell with high homologous recombination efficiency,    causing deletion and/or translocation to occur at the marked site of    said foreign chromosome(s) or a fragment(s) thereof.-   2. The method of item 1, wherein a plurality of said cells with high    homologous recombination efficiency are subjected to whole cell    fusion after steps (a) and (b) and are subjected to step (c).-   3. The method of item 2, wherein a plurality of said cells with high    homologous recombination efficiency each comprises a distinct    foreign chromosome(s) or a fragment(s) thereof.-   4. The method of item 1, wherein said cell comprising a modified    foreign chromosome(s) or a fragment(s) thereof is an animal cell.-   5. The method of item 4, wherein said animal cell is a mammalian    cell.-   6. The method of item 4, wherein said animal cell is a non-human    animal cell.-   7. The method of item 1, wherein said targeting vector contains a    telomere sequence which is introduced into a desired site by    insertion of the targeting vector.-   8. The method of item 2, wherein said deletion occurs at a site into    which said telomere sequence has been introduced.-   9. The method of item 1, wherein said targeting vector comprises a    recognition sequence for a site-directed recombination enzyme, and    said recognition sequence is introduced into a desired site by    insertion of the targeting vector.-   10. The method of item 9, wherein a vector, which is capable of    expressing a site-directed recombination enzyme, is introduced into    said cell with high homologous recombination efficiency    simultaneously with or after insertion of said targeting vector    comprising the recognition sequence for a site-directed    recombination enzyme, so that an activity of said site-directed    recombination enzyme is expressed, resulting in deletion and/or    translocation of said foreign chromosome(s) or a fragment(s) thereof    at a site in which said recognition sequence for a site-directed    recombination enzyme is introduced.-   11. The method of item 10, wherein said translocation occurs between    a plurality of foreign chromosomes or fragments thereof.-   12. The method of item 11, wherein a plurality of said foreign    chromosomes are derived from the same species.-   13. The method of item 12, wherein said same species is a human.-   14. The method of item 11, wherein a plurality of said foreign    chromosomes are derived from different species.-   15. The method of item 14, wherein said species are a human and a    mouse.-   16. The method of item 10, wherein said translocation occurs between    a foreign chromosome(s) or a fragment(s) thereof and a chromosome(s)    derived from said cell with high homologous recombination    efficiency.-   17. The method of item 9, wherein said site-directed recombination    enzyme is a Cre enzyme.-   18. The method of item 9, wherein said recognition sequence for a    site-directed recombination enzyme is a LoxP sequence.-   19. The method of item 1, wherein said cell with high homologous    recombination efficiency is an embryonic stem cell (or ES cell).-   20. The method of item 1, wherein said cell with high homologous    recombination efficiency is a chicken DT-40 cell.-   21. The method of item 1, which further comprises a step of    screening a cell comprising said foreign chromosome(s) or a    fragment(s) thereof in which deletion and/or translocation has    occurred.-   22. The method of item 21, wherein said screening is based on    expression of a marker gene.-   23. The method of item 22, wherein said marker gene is a    drug-resistance gene.-   24. The method of item 22, said marker gene is a green fluorescent    protein-encoding gene derived from the jellyfish Aequorea victoria    or a modified gene thereof.-   25. The method of item 1, wherein said foreign chromosome(s) or a    fragment(s) thereof is derived from a human.-   26. A method for producing a chimeric non-human animal comprising a    modified foreign chromosome(s) or a fragment(s) thereof, which    comprises the steps of:-   (a) preparing a microcell comprising a foreign chromosome(s) or a    fragment(s) thereof, and transferring said foreign chromosome(s) or    a fragment(s) thereof into a cell with high homologous recombination    efficiency through its fusion with said microcell;-   (b) in said cell with high homologous recombination efficiency,    inserting a vector by homologous recombination into a desired site    of said foreign chromosome(s) or a fragment(s) thereof, and/or a    desired site of a chromosome(s) derived from said cell with high    homologous recombination efficiency, thereby marking said desired    site;-   (c) in said cell with high homologous recombination efficiency,    causing deletion and/or translocation to occur at the marked site of    said foreign chromosome(s) or a fragment(s) thereof; and-   (d) preparing a microcell comprising said foreign chromosome(s) or a    fragment(s) thereof in which deletion or translocation has occurred,    and transferring said foreign chromosome(s) or a fragment(s) thereof    into a pluripotent non-human animal cell through its Fusion with    said microcell.-   27. The method of item 26, wherein a plurality of said cells with    high homologous recombination efficiency are subjected to whole cell    fusion after steps (a) and (b) and are subjected to step (c).-   28. The method of item 27, wherein a plurality of said cells with    high homologous recombination efficiency each comprise a distinct    foreign chromosome(s) or a fragment(s) thereof.-   29. The method of item 26, wherein said targeting vector comprises a    telomere sequence which is introduced into a desired site by    insertion of the targeting vector.-   30. The method of item 29, wherein said deletion occurs at a site    where said telomere sequence has been introduced.-   31. The method of item 26, wherein said targeting vector comprises a    recognition sequence for a site-directed recombination enzyme, and    said recognition sequence is introduced into a desired site by    insertion of the targeting vector.-   32. The method of item 31, wherein a vector, which is capable of    expressing a site-directed recombination enzyme, is introduced into    said cell with high homologous recombination efficiency    simultaneously with or after insertion of said targeting vector    comprising said recognition sequence for a site-directed    recombination enzyme, so that an activity of said site-directed    recombination enzyme is expressed, resulting in deletion and/or    translocation of said foreign chromosome(s) or a fragment(s) thereof    at a site into which said recognition sequence is introduced.-   33. The method of item 32, wherein said translocation occurs between    a plurality of foreign chromosomes or fragments thereof.-   34. The method of item 32, wherein said translocation occurs between    said foreign chromosome(s) or a fragment(s) thereof and said    chromosome(s) derived from said cell with high homologous    recombination efficiency.-   35. The method of item 31, wherein said site-directed recombination    enzyme is a Cre enzyme.-   36. The method of item 31, wherein said recognition sequence for    site-directed recombination enzyme is a LoxP sequence.-   37. The method of item 26, wherein said cell with high homologous    recombination efficiency is an embryonic stem cell (or ES cell).-   38. The method of item 26, wherein said cell with high homologous    recombination efficiency is a chicken DT-40 cell.-   39. The method of item 26, which further comprises a step of    screening cells comprising a foreign chromosome(s) or a fragment(s)    thereof in which deletion and/or translocation has occurred.-   40. The method of item 39, wherein said screening is based on    expression of a marker gene.-   41. The method of item 40, wherein said marker gene is a    drug-resistance gene.-   42. The method of item 40, the marker gene is a green fluorescent    protein-encoding gene derived from the jellyfish Aequorea victoria    or a modified gene thereof.-   43. The method of item 26, wherein in the step (d), a microcell is    produced from said cell with high homologous recombination    efficiency; said foreign chromosome(s) or a fragment(s) thereof, in    which deletion and/or translocation has occurred is transferred into    a CHO cell through its fusion with said Microcell; a microcell is    produced from the CHO cell; and then said foreign chromosome(s) or a    fragment(s) thereof in which deletion and/or translocation has    occurred is transferred into a pluripotent cell through its fusion    with said microcell.-   44. The method of item 26, said pluripotent cell is an embryonic    stem cell (or ES cell).-   45. The method of item 26, said foreign chromosome(s) or a    fragment(s) thereof is derived from a human.-   46. A method for producing a non-human animal comprising a modified    foreign chromosome(s) or a fragment(s) thereof, which comprises the    steps of:-   (a) preparing a microcell comprising a foreign chromosome(s) or a    fragment(s) thereof, and transferring said foreign chromosome(s) or    a fragment(s) thereof into a cell with high homologous recombination    efficiency through its fusion with said microcell;-   (b) in said cell with high homologous recombination efficiency,    inserting a vector by homologous recombination into a desired site    of said foreign chromosome(s) or a fragment(s) thereof, and/or a    desired site of a chromosome(s) derived from said cell with high    homologous recombination efficiency, thereby marking said desired    site;-   (c) in said cell with high homologous recombination efficiency,    causing deletion and/or translocation to occur at the marked site of    said foreign chromosome(s) or a fragment(s) thereof;-   (d) preparing a microcell comprising said foreign chromosome(s) or a    fragment(s) thereof, in which deletion and/or translocation has    occurred, and transferring said foreign chromosome(s) or a    fragment(s) thereof into a cell derived from a non-human animal    through its fusion with said microcell; and-   (e) transplanting the nucleus of said cell derived from the    non-human animal into an enucleated unfertilized egg derived from a    homologous non-human animal of the same species.-   47. The method of item 46, wherein a plurality of said cells with    high homologous recombination efficiency are subjected to whole cell    fusion after steps (a) and (b) and are subjected to the step (c).-   48. The method of item 47, wherein a plurality of said cells with    high homologous recombination efficiency comprise a distinct foreign    chromosome(s) or a fragment(s) thereof.-   49. The method of item 46, wherein said targeting vector comprises a    telomere sequence, which is introduced into a desired site by    insertion of the targeting vector.-   50. The method of item 49, wherein said deletion occurs at a site    into which a telomere sequence has been introduced.-   51. The method of item 46, wherein said targeting vector comprises a    recognition sequence for site-directed recombination enzyme, and    said recognition sequence is introduced into a desired site by    insertion of the targeting vector.-   52. The method of item 51, wherein a vector, which is capable of    expressing a site-directed recombination enzyme, is introduced into    said cell with high homologous recombination efficiency    simultaneously with or after insertion of said targeting vector    comprising said recognition sequence for a site-directed    recombination enzyme, so that an activity of said site-directed    recombination enzyme is expressed, resulting in deletion and/or a    translocation of said foreign chromosome(s) or fragment(s) thereof    at a site into which said recognition sequence is introduced.-   53. The method of item 52, wherein said translocation occurs between    a plurality of foreign chromosomes or fragments thereof.-   54. The method of item 52, wherein said translocation occurs between    said foreign chromosome(s) or a fragment(s) thereof and said    chromosome derived from a cell with high homologous recombination    efficiency.-   55. The method of item 51, wherein said site-directed recombination    enzyme is a Cre enzyme.-   56. The method of item 51, wherein said recognition sequence for a    site-directed recombination enzyme is a LoxP sequence.-   57. The method of item 46, wherein said cell with high homologous    recombination efficiency is an embryonic stem cell (or ES cell).-   58. The method of item 46, wherein said cell with high homologous    recombination efficiency is a chicken DT-40 cell.-   59. The method of item 46, which further comprises a step of    screening cells containing a foreign chromosome(s) or a fragment(s)    thereof in which deletion and/or translocation has occurred.-   60. The method of item 59, wherein said screening is based on    expression of a marker gene.-   61. The method of item 60, wherein said marker gene is a    drug-resistant gene.-   62. The method of item 60, wherein said marker gene is a green    fluorescent protein-encoding gene derived from the jellyfish    Aequorea victoria or a modified gene thereof.-   63. The method of item 46, wherein, in the step (d), a microcell is    produced from said cell with high homologous recombination    efficiency; said foreign chromosome(s) or fragment(s) thereof, in    which deletion and/or translocation have/has occurred, is/are    transferred into a CHO cell through its fusion with the microcell; a    microcell is produced from the CHO cell; and then said foreign    chromosome(s) or a fragment(s) thereof, in which deletion and/or    translocation has occurred, is transferred into a cell derived from    a non-human animal through its fusion with the microcell.-   64. The method of item 46, said cell derived from a non-human animal    is a culture cell derived from an embryo or a blastocyst.-   65. The method of item 46, said cell derived from a non-human animal    is a culture cell derived from a fetus or an adult.-   66. The method of item 46, said cell derived from a non-human animal    is a fibroblast cell derived from fetus.-   67. The method of item 46, said foreign chromosome(s) or a    fragment(s) thereof is derived from a human.-   68. A non-human animal, which retains a chromosomal fragment(s)    obtained by deletion of a foreign chromosome(s) or a fragment(s)    thereof.-   69. The non-human animal of item 68, wherein said chromosomal    fragment(s) comprises:    (i) a marker gene and a telomere sequence, and/or    (ii) a recognition sequence for a site-directed recombination    enzyme.-   70. A non-human animal, comprising a recombinant foreign    chromosome(s) obtained by translocation between a plurality of    foreign chromosomes or fragments thereof.-   71. The non-human animal of item 70, wherein said recombinant    chromosomal fragment(s) comprises:    (i) a marker gene and a telomere sequence; and/or    (ii) a recognition sequence for a site-directed recombination    enzyme.-   72. The non-human animal of item 70, wherein said recombinant    foreign chromosome(s) is independently maintained in the nucleus of    the non-human animal cell.-   73. The non-human animal of item 70, wherein said recombinant    foreign chromosome(s) is derived from a human.-   74. The non-human animal of item 70, wherein the recombinant foreign    chromosome(s) is derived from human chromosomes #14 and #2.-   75. The non-human animal of item 70, wherein said recombinant    foreign chromosome(s) is derived from human chromosomes #14 and #22-   76. The non-human animal of item 70, wherein said recombinant    foreign chromosome(s) comprises genes for a heavy-chain and a    light-chain λ of a human antibody.-   77. The non-human animal of item 70, wherein said recombinant    foreign chromosome(s) comprises genes for a heavy-chain and a    light-chain κ gene of a human antibody.-   78. The non-human animal of item 70, which is a mouse.-   79. The non-human animal of item 70, which is an ungulata.-   80. The non-human animal of item 70, which is a bovine.-   81. The non-human animal of item 70, which is an ovine.-   82. The non-human animal of item 70, which is an avian.-   83. The non-human animal of item 70, which is a chicken.-   84. A cell, comprising a recombinant chromosome(s) or a fragment(s)    thereof obtained by deletion and/or translocation of a chromosome(s)    or a fragment(s) thereof, which comprises at least a part of a human    chromosome and into which    (i) a marker gene and telomere sequence, and/or    (ii) a recognition sequence for site-directed recombination enzyme    is/are introduced.-   85. A method for modifying a foreign chromosome(s) or a fragment(s)    thereof in a cell, which comprises the steps of:-   (a) preparing a microcell containing a foreign chromosome(s) or a    fragment(s) thereof, and transferring said foreign chromosome(s) or    a fragment(s) thereof into a cell with high homologous recombination    efficiency through its fusion with the microcell;-   (b) in said cell with high homologous recombination efficiency,    inserting a targeting vector by homologous recombination into a    desired site of said foreign chromosome(s) or a fragment(s) thereof    and/or a desired site of a chromosome(s) derived from said cell with    high homologous recombination efficiency, thereby marking said    desired site; and-   (c) in said cell with high homologous recombination efficiency,    causing deletion or translocation to occur at the marked site of    said foreign chromosome(s) or a fragment(s) thereof.-   86. An artificial chromosome vector, which comprises a centromere    sequence derived from a human chromosome #14 or #21, and a    recognition sequence for a site-directed recombination enzyme.-   87. The artificial chromosome vector of item 86, wherein said    recognition sequence for a site-directed recombination enzyme is a    LoxP sequence.-   88. A recombinant chromosome or a fragment thereof obtained by    deletion and/or translocation of a chromosome(s) or a fragment(s)    thereof, into which    (i) a marker gene and a telomere sequence, and/or    (ii) a recognition sequence for a site-directed recombination enzyme    is/are introduced, and which comprises at least a part of a human    chromosome.-   89. The recombinant chromosome or a fragment thereof of item 88,    which comprises fragments of human chromosomes #14 and #22.-   90. The recombinant chromosomes or a fragment thereof of item 88,    which comprises fragments of human chromosomes #14 and #2.-   91. The recombinant chromosomes or a fragment thereof of item 88,    which comprises genes for a heavy-chain and a light-chain λ of a    human antibody.-   92. The recombinant chromosomes or a fragment thereof of item 88,    which comprises genes for a heavy-chain and a light-chain κ of a    human antibody.

The present specification includes the contents as disclosed in thespecification and/or drawings of Japanese Patent Application No.10-236169, which is a priority document of the present application.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of PCR analysis of an A9 cell retaining humanchromosome #2 (fragment).

FIG. 2 shows that human chromosome #22 (fragment) is retained in an E14drug resistant cell (PCR analysis).

FIG. 3 is a photograph of electrophoresis patterns showing that human L1sequence is retained in a chimeric mouse produced from a humanchromosome #22-transferred ES cell (Southern analysis).

FIG. 4 is a photograph of electrophoresis patterns showing the presenceof a human chromosome in organs of a human chromosome #22 transferredchimeric mouse (PCR analysis).

FIG. 5 is a photograph of electrophoresis patterns showing the resultsof the expression of human genes in a human chromosome #22 transferredchimeric mouse (RT-PCR).

FIG. 6 is a photograph of electrophoresis patterns showing the resultsof the expression of human genes in organs of a human chromosome #22transferred chimeric mouse (RT-PCR)

FIG. 7 shows that human chromosome #4 (fragment) is retained in an E14drug resistant cell (PCR analysis).

FIG. 8 is a photograph of electrophoresis patterns showing the detectionof human L1 sequence in a human chromosome #4-transferred E14 cell clone(Southern analysis).

FIG. 9 is a photograph of electrophoresis patterns showing that human L1sequence is retained in a chimeric mouse produced from a humanchromosome #4-transferred ES cell (Southern analysis).

FIG. 10 shows that human chromosome #14 (fragment) is retained in a TT2drug resistant cell (PCR analysis).

FIG. 11 is a photograph of electrophoresis patterns showing the presenceof a human chromosome in organs of a chimeric mouse produced from ahuman chromosome #14 transferred ES cell (PCR analysis).

FIG. 12 shows the results of a test on a tail-derived fibroblast cellfor resistance to G418.

FIG. 13 shows the concentration of human antibody IgM in a serum of ahuman serum albumin (hereinafter referred to as “HSA”)-immunizedchimeric mouse (ELISA).

FIG. 14 shows the concentration of human antibody IgG in a serum of anHSA-immunized chimeric mouse (ELISA)

FIG. 15 shows the results of ELISA of hybridoma clone H4B7 capable ofproducing human IgM.

FIG. 16 is a photograph of the results of FISH analysis of a mouse EScell clone (TT2 cell clone PG15) retaining partial fragments of humanchromosomes #2 and 14.

FIG. 17 shows that the antibody titer of anti-HSA human IgG is increasedin a serum of an HSA-immunized chimeric mouse.

FIG. 18 shows that the antibody titer of anti-HSA human Igκ is increasedin a serum of an HSA-immunized chimeric mouse.

FIG. 19 is a photograph of electrophoresis patterns showing thedetection of human L1 sequence in a human chromosome #22-transferred TT2cell clone (Southern analysis).

FIG. 20 shows that the antibody titer of anti-HSA human Igλ is increasedin a serum of an HSA-immunized chimeric mouse.

FIG. 21 is a photograph showing that a partial fragment of humanchromosome #2 is retained in a progeny of a chimeric mouse into which apartial fragment of a human chromosome #2 was transferred (PCRanalysis).

FIG. 22 shows the presence of a cell expressing human μ chain on thecell surface in a spleen of a human chromosome #14-transferred chimericmouse (flow-cytometry analysis).

FIG. 23 shows the structure of LoxP-pstNEO plasmid DNA.

FIG. 24 shows the structure of genomic DNA carrying a mouse antibodyheavy chain Cμ gene.

FIG. 25 shows the structure of genomic DNA carrying a mouse antibodylight-chain κ gene.

FIG. 26 shows the structures of a mouse antibody heavy-chain targetingvector and a probe for Southern blotting, as well as a DNA fragment tobe detected in homologous recombinants.

FIG. 27 shows the structures of a mouse antibody light-chain κ targetingvector and a probe for Southern blotting, as well as a DNA fragment tobe detected in homologous recombinants.

FIG. 28 is a photograph of electrophoresis patterns showing the resultsof Southern blot analysis of mouse antibody heavy-chain homologousrecombinants and high concentration G418 resistant clones derivedtherefrom.

FIG. 29 shows a photograph of electrophoresis patterns showing theresults of Southern blot analysis of mouse antibody light-chainhomologous recombinants.

FIG. 30 shows the structure of pLoxP-PGKPuro plasmid DNA.

FIG. 31 shows a mouse antibody light-chain K targeting vector, a probefor use in the southern blot analysis of genomic DNA from transformantTT2F cells, and DNA fragments to be detected in homologous recombinants.

FIG. 32 shows a photograph of electrophoresis patterns showing theresults of Southern blot analysis of high concentration G418 resistantcell clones derived from mouse antibody light-chain homologousrecombinants.

FIG. 33 shows that the antibody titers of anti-HSA human IgH antibodiesare increased in a serum of an HSA-immunized chimeric mouse.

FIG. 34 is a photograph of the result of FISH analysis of an antibodyheavy- and light-chains deficient mouse ES cell clone retaining partialfragments of human chromosomes #2 and #14.

FIG. 35 shows that the antibody titers of anti-HSA human Ig antibodiesare increased in a serum of an HSA-immunized chimeric mouse.

FIG. 36 is a photograph showing the result of FISH analysis of a mouseA9 cell containing human chromosome #14 (human centromere sequenceprobe).

FIG. 37 is a photograph showing the result of FISH analysis of a mouseA9 cell containing human chromosome #14 (human chromosome-specificprobe).

FIG. 38 shows the results of a test for stability of human chromosomefragments (#14: SC20, #2:W23) in a mouse ES cell.

FIG. 39 shows the results of analysis for stability of human chromosome#14 fragments in a mouse.

FIG. 40 shows the results of PCR analysis of a G418 resistant hybridcells retaining human chromosome #22 (fragment).

FIG. 41 is a photograph showing the results of FISH analysis of an A9cell retaining fragmented human chromosome #22.

FIG. 42 shows the results of complete human antibody-producing mousestrains established by mating.

FIG. 43 shows the results of the determination of the concentration ofhuman antibody κ chain in a serum of a mouse retaining a humanchromosome #2 fragment, W23.

FIG. 44 shows the results of the determination of the concentration ofhuman antibody κ and λ chains in a serum of a mouse.

FIG. 45 shows the structure of pBS-TEL/LIFPuro.

FIG. 46 shows that human chromosome #22 is retained in a chicken DT40cell clone.

FIG. 47 shows the identification of homologous recombinant in LIF locus.

FIG. 48 shows the fragmentation of human chromosome #22 in a DT40/#22neocell clone.

FIG. 49 is a photograph showing a chicken DT40 cell clone retaining fulllength or fragmented human #22 chromosome.

FIG. 50 shows an increase in the anti-TNF-α human Igγ antibody titer inthe sera of TNF-α-immunized chimeric mice.

FIG. 51 shows the response to ASIALO-GM1 in chimeric mice.

FIG. 52 shows the response to GM2 in chimeric mice.

FIG. 53 shows the results of FACS analysis for single Tc/KO mouseperipheral blood nuclear cells.

FIG. 54 shows the results of measurements of human Ig concentration inthe sera of single Tc/KO mice.

FIG. 55 shows the results of measurements of human Igγ subclassconcentration in the sera of single Tc/KO mice.

FIG. 56 shows a rise in the anti-HSA human Igγ antibody titer in thesera of HSA-immunized double Tc/KO mice.

FIG. 57 shows a rise in the anti-HSA human Igκ antibody titer in thesera of HSA-immunized double Tc/KO mice.

FIG. 58 shows the outline of the production of human artificialchromosomes λ-HAC and κHAC.

FIG. 59 shows the outline of production of human artificial chromosomesλ-HAC and κ-HAC-introduced mice.

FIG. 60 shows the structure of cassette vectors ploxPHyg and ploxPbsr.

FIG. 61 shows the structure of a targeting vector pHCF2loxPHyg(F) and amethod for identifying a homologous recombinant.

FIG. 62 shows the structure of a targeting vector pHCF21oxPHyg(R) and amethod for identifying a homologous recombinant.

FIG. 63 shows the structure of a targeting vector pRNR21oxPbsr and amethod for identifying a homologous recombinant.

FIG. 64 shows the structure of a targeting vector pYHZ1oxPHyg(F) and amethod for identifying a homologous recombinant.

FIG. 65 shows the structure of a cassette vector pTELPuro.

FIG. 66 shows the structure of a targeting vector pTELPuroCD8A(F) and amethod for identifying a homologous recombinant.

FIG. 67 shows the structure of a targeting vector pTELPuroCD8A(R) and amethod for identifying a homologous recombinant.

FIG. 68 shows the presence of a genetic marker on human chromosome 2 andthe presence of a genetic marker on human chromosome 2 in a clone CD10in which telomere was truncated at CD8A locus.

FIG. 69 shows the results of FISH for the clone CD10 using a pGKPuroprobe.

FIG. 70 shows the structure of a vector, pBS185hisD, for stableexpression of Cre recombination enzyme.

FIG. 71 shows the structure of an artificial chromosome aftertranslocation between loxPs of RHF clone and of RHR clone.

FIG. 72 shows PCR identification for confirming the structure of agenomic region after translocation between loxP in RHF clone andconfirming the translocation.

FIG. 73 shows the results of PCR for confirming translocation.

FIG. 74 shows the results of FISH for RHF clone using a pGKPuro probeand a 14qter specific probe, and results of FISH for the same cloneusing a chromosome 22-specific probe and a chromosome 14-specific probe.

FIG. 75 shows a rise in anti-HSA human Igγ antibody titer and in Igλantibody titer in the sera of HAS-immunized chimeric mice C-10.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described in detail.

A mouse that retains a human chromosome(s) or a fragment(s) thereof andwhich expresses the gene on the chromosome(s) or fragment(s) thereof canbe produced by

(1) preparing a chromosome donor cell which retains a labeled humanchromosome or a fragment thereof;(2) transferring the human chromosome or fragment thereof into a murinepluripotent cell by microcell fusion;(3) producing a chimeric mouse from the cell; and(4) confirming that the human chromosome is retained in the chimericmouse and that a human gene is expressed.

In this procedure, a mouse is used as a non-human animal that retains ahuman chromosome or a fragment thereof and which expresses the gene onthe chromosome or fragment thereof (the mouse is hereinafter referred toas a “human chromosome transferred mouse”).

The term “human chromosome” means a naturally occurring complex whichconsists of nucleic acids and proteins that are derived from humancells. There are 46 normal human chromosomes of 23 kinds (24 kinds inmale), each of which contains DNAs of about 50-300 Mb in size. In thepresent invention, the human chromosome includes not only partialfragments which can be stably replicated and segregated as independentchromosomes but also fragments that are translocated on mousechromosomes and which are retained stably. The size of the DNA isusually at least 1 Mb and in some cases, it is smaller than 1 Mb. Thefeature of the present invention resides in that a mouse can retain andexpress the foreign gene on a foreign chromosome as a mediator withouttreatments such as cloning in an E. coli or yeast cell, or extraction ofthe DNA from a cell.

The term “human chromosome transferred mouse” means a mouse retaining ahuman chromosome(s) or a fragment(s) thereof in all or part of itsnormal somatic cells. The mouse expresses the gene(s) on a humanchromosome(s) or a fragment(s) thereof in all or part of its normalsomatic cells.

(1) Preparation of a Chromosome Donor Cell which Retains a Labeled HumanChromosome or a Fragment Thereof

A desired chromosome donor cell 1) retains a human chromosome(s) labeledwith a marker also available for selection of recipient cells; 2) doesnot contain other human chromosomes; and 3) has a higher ability to forma microcell.

Any human-derived cell lines, cancer cells and primary culture cells canbe used as materials for providing human chromosomes. Among them, normalfibroblast cells are suitable because they have a low possibility ofabnormality such as deletion and amplification of chromosomes and can bereadily cultured.

As for 1), human cells can be transformed with vectors that expressgenes for markers such as drug-resistance (e.g., G418-, puromycin-,hygromycin- or blasticidin-resistance). Promoters operating efficientlynot only in human cells but also in recipient cells such as mouse EScells are desirably used to regulate the expression of the marker used.For this purpose, herpes simplex virus thymidine kinase promoter linkedwith SV 40 enhancer (Katoh et al., Cell Struct. Funct., 12:575, 1987),mouse PGK-1 promoter (Soriano et al., Cell, 64:693, 1991) and the likecan be used. A library of human cell transformants in which theintroduced marker genes have been inserted into 46 human chromosomes of23 kinds at random can be prepared by transformation throughelectroporation (Ishida et al., “Cell Technology Experiment Manual”,published by Kodansha, 1992) and the like and subsequent selection oftransformants.

As for 3), since many human normal cells have a very low ability to formmicrocells, the whole cell of the transformant may be fused with a cellhaving a high ability to form microcells such as mouse A9 cell(Oshimura, M., Environ. Health Perspect., 93:57, 1991) so as to providethe transformed cell with an ability to form microcells. It is knownthat in mouse-human hybrid cells, human chromosomes selectivelydisappear. The fused cell selected by the marker can retain stably themarked human chromosome.

In order to meet the condition of 2), it is desired to obtain amicrocell from the fused cell and fuse it again with a mouse A9 cell. Inthis case, too, most of the cells selected by the marker will meet thethree conditions 1), 2) and 3) above. The marked human chromosomes canbe identified in the finally obtained mouse-human monochromosomal hybridcells by PCR (Polymerase Chain Reaction, Saiki et al., Science, 239:487,1988), Southern blot analysis (Ausubel et al., Current protocols inmolecular biology, John Wiley & Sons, Inc., 1994), FISH analysis(Fluorescence In Situ Hybridization, Lawrence et al., Cell, 52:51, 1988)and the like. If the transfer of a specified chromosome is desired, theabove procedures are applied to each of many human cell transformantclones to select a clone in which a chromosome of interest is marked.Alternatively, the above procedures are applied to a mixture of humancell transformant clones and the identification of human chromosomes iscarried out on a large number of the resulting mouse-humanmonochromosome hybrid cells.

In addition, a marker gene can be inserted into a desired site byhomologous recombination of a specific DNA sequence on the chromosomewhich is to be transferred (Thomas et al., Cell, 51:503, 1987).

A microcell prepared from the mouse-human hybrid cell may be irradiatedwith γ-rays such that the marked human chromosome is fragmented andtransferred into a mouse A9 cell. Even if the microcell is notirradiated with γ-rays, a partially fragmented human chromosome may betransferred at a certain frequency. In these cases, the resultingmicrocell fused clones retain partial fragments of the marked humanchromosomes. These clones can be used when it is desired to transfer thepartial fragments into recipient cells.

Human chromosomes to be introduced into ES cells may be modified bydeletion, translocation, substitution and the like. Specific proceduresfor these modifications are as follows:

1) In each of the steps of preparing the aforementioned mouse-humanhybrid cell, inducing a microcell from the mouse-human hybrid cell,further fusing the microcell with a mouse A9 cell, inducing a microcellfrom the further fused cell and fusing the latter microcell with a mouseES cell, deletion and/or translocation of human chromosomes mayoccasionally occur. Cells retaining such mutated chromosomes areselected under the microscopic observation of chromosomes or by use ofPCR, Southern analysis, or the like. A clone retaining a desired mutantchromosome can be selected from a mouse A9 library retaining varioushuman chromosomes. A clone retaining a desired mutant chromosome can beselected from A9 or ES cell fused with a microcell induced from a mouseA9 cell retaining a certain human chromosome. The frequency offragmentation of chromosomes can be raised by γ-ray irradiation (Koi etal., Science, 260:361, 1993).2) A targeting vector retaining a loxp sequence that is recognized byCre enzyme is constructed. A clone into which a loxp sequence has beeninserted at a desired site on a chromosome is obtained by homologousrecombination in a cell retaining a human chromosome. Subsequently, Creenzyme is expressed in the cell of the clone to select a mutant havingchromosomal deletion and/or translocation caused by site-directedrecombination. See WO97/49804 and Smith et al., Nature Genetics, 9:376,195. As a host into which a targeting vector is to be introduced, a cellallowing for high-frequency homologous recombination such as a chickenDT40 cell (Dieken et al., Nature Genetics, 12:174, 1996) may also beused.3) A targeting vector retaining a human telomere sequence is constructedand the telomere sequence is inserted in the cell at a desired site on achromosome by homologous recombination in a cell retaining a humanchromosome. After a clone into which the telomere sequence has beeninserted is obtained, a mutant having deletion caused by the telomeretruncation is obtained. See Itzhaki et al., Nature Genet., 2, 283-287,1992 and Brown et al., P. N. A. S., 93:7125, 1996. As a host into whicha targeting vector is to be introduced, a cell allowing forhigh-frequency homologous recombination such as a chicken DT40 cell(Dieken et al., supra) may also be used. Telomere truncation of humanchromosomes in a chicken DT40 cell is first disclosed in the presentinvention Brown (supra) discloses that a vector was inserted into arepeat sequence on a chromosome. However, no specific site can betargeted. Itzhaki et al. discloses that tumor cells, i.e., 12000 cellsof cell line HT1080 into which a telomere sequence was introduced wereanalyzed and 8 homologous recombinants were obtained. They found thatout of the 8 cells, only one caused deletion by insertion of thetelomere sequence. For some kinds of cells, results were reported thatno mutant having truncation was obtained by insertion of a telomeresequence into some kinds of cells (Barnett et al., Nucleic Acids Res.,21:27, 1993). In spite of this report, the inventors believed that itwas necessary to increase the absolute number of homologous recombinantsin order to obtain mutants having truncation and made an attempt toperform telomere truncation using a chicken DT40 cell as a host. As aresult, it was surprisingly found that truncation occurred in all of the8 homologous recombinants obtained.

As mentioned above, a gene that should not be expressed in a humanchromosome-transferred mouse can be removed by modification of aintroduced chromosome. If the size of a chromosome to be transferred isshortened by fragmentation, the chromosome fragment to be transferredcan be transmitted to progenies of the chromosome-transferred mice. Inaddition, using chromosome translocation and substitution techniques,genes derived from a plurality of chromosomes can be expressed on thesame chromosome fragment and portions of a plurality of genes on thechromosome fragments can be replaced with different genes. In otherwords, foreign chromosome fragments can be used as vectors fortransferring genes into individual mice and their cells.

A more detailed description of a method for artificially modifying humanchromosomes will now be given.

To date, an accidentally fragmented human chromosome or a full lengthhuman chromosome has been introduced into a mouse (Tomizuka et al.,Nature Genet., 16: 133, 1997). In this case, some of the introducedhuman chromosomes may be unstable in a mouse, or may affect developmentof the mouse so that a human chromosome-introduced mouse itself cannotbe generated. Furthermore, it is assumed that an introduced humanchromosome cannot be transmitted to its progeny since the presence of anabnormal chromosome in a human or a mouse is thought to inhibit meiosis(Tomizuka et al., Nature Genet., 16: 133, 1997). In fact, Tomizuka etal. reported that male chimeric mice retaining a fragment which containsa region approximately half a human chromosome 14 (about 100 Mb) becameinfertile, and this fragment was not transmitted to progeny (NatureGenet., 16: 133, 1997). On the other hand, small chromosomal fragments,such as a fragment W23 of chromosome 2, (it is thought to be 5 to 20 Mb;Rastan, Nature Genet., 16: 113, 1997) as described in Tomizuka et al.'sreport and a fragment SC20 of chromosome 14, (somewhat larger than W23)as shown in Examples of the specification, could be transmitted toprogeny. That is, the use of smaller chromosomal fragments may increasethe possibility for them to be transmitted to progeny.

In order to produce a mouse, which contains a target region of a humanchromosome, stably retains it, and then transmits it to the progeny,there is a need to develop techniques which make it possible to freelyprocess a chromosome. That is, techniques that do not depend onaccidentally obtained chromosomal fragments, but enables us to cleave ahuman chromosome at a desired site or to combine only a desiredchromosomal fragment with another stable chromosome. These techniquesare called chromosome engineering, and have been used to date forgenerating a mutant mouse having a modified chromosome, by site-directedcleavage of an endogenous mouse chromosome mainly in a mouse ES cell (Wo98/54348), or by causing recombination (translocation) betweenhomologous chromosomes so as to cause deletion, inversion, ormultiplication of a specific genetic region (R. Ramirez-Solis et al.,Nature 378: 720, 1995). There have been no reports on production of anon-human animal, such as a mouse, having a human chromosome(s)artificially modified in these various ways. Recently, it has beenreported that an artificial chromosome vector (Mammalian ArtificialChromosome, MAC), which is stably retained in a mammalian cell, has beendeveloped (for example, W. Mills et al., Human Molecular Genetics 8:751, 1999). However, there has been no report on a universalconstruction of an artificial chromosome, which allows cloning of agenomic region exceeding the cloning size of YAC vector.

This invention provides a universal system for constructing humanartificial chromosomes, which will allow cloning of any chromosomalfragments showing production of a human artificial chromosome (HAC) intowhich a human antibody gene cluster is cloned as an example.

The production of human artificial chromosomes λ-HAC and κ-HAC havinghuman antibody λ light-chain and κ light-chain gene clusters introducedthereto will be described as follows. It will include the introductionof the produced HAC into a mouse, expression of human antibody genescontained in HAC in a mouse, and transmission of HAC to the progeny ofchimeric mice.

A. Modification of Human Chromosome 22

A human antibody λ light-chain gene cluster is present at 22q11.2 onchromosome 22 (for example, J. E. Collins et al., Nature 377: 367,1995). To use only the periphery of this region of the chromosome forproduction of an artificial chromosome, first, a human telomere sequenceis inserted by a homologous recombination into LIF locus closely linkedto a λ light-chain gene cluster in the telomere side resulting intelomere-directed truncation to cleave the chromosome (for example,Kuroiwa et al., Nucleic Acid Research, 26: 3447, 1998). Next, arecognition sequence, loxP sequence, of a recombination enzyme, Cre, isinserted by homologous recombination into the HCF2 locus closely linkedto the λ light-chain gene cluster in the centromere side. Here, onlyHCF2-Igλ-LIF fragments are used for the production of artificialchromosomes. By cloning only fragments of chromosome 22, causativegenetic regions on the chromosome 22 of Cats Eye syndrome (for example,Johnson. A et al., Genomics 57: 306, 1999) and Digeorge syndrome (forexample, H. Yamagishi et al., SCIENCE 283: 1158, 1999), which may affectthe development of a mouse, can be eliminated.

B. Modification of Human Chromosome 2

A human antibody κ light-chain gene cluster is present at 2p11.2 onchromosome 2 (for example, Gottfried M. et al., Genomics 16: 512, 1993).To use only the periphery of this region of the chromosome forproduction of an artificial chromosome, first, a human telomere sequenceis inserted by homologous recombination into CD8A locus (for example,Gottfried M. et al., Genomics 16: 512, 1993) closely linked to the κlight-chain gene cluster in the telomere side. This insertion results intelomere-directed truncation to cleave the chromosome. Next, arecognition sequence, loxP sequence, of a recombination enzyme, Cre, isinserted by homologous recombination into the cosYHZ304 genomic regionclosely linked to the κ light-chain gene cluster in the centromere side.Here, only cosYHZ304-Igκ-CD8A fragment is used for production ofartificial chromosomes.

C. Modification of Human Chromosome 14 Fragment SC20

It has been shown that human chromosome 14 fragment SC20, is retainedalmost 100% in a mouse and can be transmitted to progeny. Therefore, inthis invention, SC20 is used as a human artificial chromosome vector.That is, the translocation of HCF2-Igλ-LIF fragment of human chromosome22 and cosYHZ304-Igκ-CD8A fragment of human chromosome 2 as describedabove in A and B to SC20 resulting in construction of human artificialchromosomes λ and κ-HAC having the fragments of the human chromosomes 22and 2 cloned thereto.

First, a loxP sequence is inserted by homologous recombination into RNR2locus located at 14p12 of chromosome 14. The Cre-loxP system (forexample, R. Ramirez-Solis et al., Nature 378:720, 1995) is applied forcloning the HCF2-Igλ-LIF fragment of human chromosome 22 andcosYHZ304-Igκ-CD8A fragment of human chromosome 2 into the RNR2 locus onSC20 by translocation.

For efficient telomere-directed truncation or homologous recombinationas described in A, B, and C above, a chicken DT-40 cell can be used as ahost cell (for example, Kuroiwa et al., nucleic Acid Research 26: 3447,1998, and Dieken et al., Nature Genet., 12: 174, 1996).

Furthermore, it was found that human chromosome 21 (fragment) screenedfrom a monochromosome hybrid cell library that has been constructed bythe methods described in Tomizuka et al.'s report (Nature Genet., 16:133, 1997) was also very stable in a chimeric mouse. That is, a chimericmouse was produced from mouse ES cells, which retain chromosomalfragments containing most of human chromosome 21 (chimerism: 95% ormore). The retention rate of the chromosome in a brain cell, a hepaticcell, and a fibroblast derived from the tail of this chimeric mouse wereall 95% or more. Hence, it is concluded that human chromosome 21 andfragments thereof can be used as a human artificial chromosome vector,as in the above SC20.

D. Translocation of HCF2-Igλ-LIF Fragment of Human Chromosome 22 andcosYHZ304-Igκ-CD8A Fragment of Human Chromosome 2 onto SC20 by Cre-loxPSystem

As described in A, B and C above, a chicken DT-40 cell, which retainseither a fragment of the modified human chromosome 22 or 2, or amodified SC20, is produced. To translocate a HCF2-Igλ-LIF fragment ofhuman chromosome 22 or cosYHZ304-Igκ-CD8A fragment of human chromosome 2onto SC20 by the Cre-loxP system, chicken DT-40 cells, each retainingeither one modified human chromosomal fragment, are fused to each other.A chicken DT-40 hybrid cell retaining two modified human chromosomalfragments is produced. In this way, all the materials required fortranslocation are prepared.

Next, the Cre recombination enzyme is expressed in the chicken DT-40hybrid cell to cause translocation. The recombination (translocation)frequency between two non-homologous chromosomes is reported to be verylow (for example, A. J. Smith et al., Nature Genet., 9:376, 1995).Moreover in this invention, recombination efficiency may be lower sincethe translocation occurs between exogenous human chromosomes, notbetween endogenous chromosomes. Accordingly, a positive selectionsystem, which can select a cell in which recombination occurs betweenloxPs as expected, must be devised.

In recent years, A. J. H. Smith et al. (Nature Genet., 9:376, 1995)succeeded in causing translocation to occur between chromosomes 12 and15 in a mouse ES cell using the Cre-loxP system. They inserted a portionon the 5′ side (including loxP) and a portion on the 3′ side (includingloxP) of the Hprt gene into specific regions of chromosomes 12 and 15 ina Hprt (hypoxanthine-guanin-phosphoribosyl transferase) gene-deficientES cell, respectively, by homologous recombination. Since a Hprt gene isre-constructed only when recombination occurs between loxPs after thetransient expression of Cre, the ES cells can grow even in a medium (HATmedium) lacking hypoxanthine. However, this selection method can be usedonly for Hprt gene-deficient cells. That is, the method cannot be usedfor all cells. As Qin, M. et al. (PNAS 91: 1706, 1994) reported, thereis a selection method, by which, instead of Hprt genes, appropriatedrug-resistant genes are reconstructed as a result of translocation, andtarget cells grow in a medium containing the drug. In this invention,the inventors have devised the following system that can be applied forany type of cell and allows rapid concentration of target cells.

In recent years, a GFP (Green Fluorescent Protein) gene (for example,Prasher D. C. et al., Gene, 111: 229, 1992) and a modified gene thereof(for example, Heim et al., Nature, 373: 663, 1995) have been developedas reporter genes for introduction of a gene into an animal cell. Sinceemission of GFP requires no substrate and GFP can be detected withfluorescence, live cells can be monitored in a short time. Even when theexpression amount of GFP is low, GFP is gradually accumulated to adetectable level in a cell because of its stability. Furthermore, theuse of FACS allows detection of very weak fluorescence. Therefore, inthis invention, a GFP gene is used as a positive selection marker forloxP recombinant. For example, a GFP gene containing no promoter isinserted together with loxP into RNR2 locus on SC20, and then a promoterrequired for the expression of GFP is inserted together with loxP intoHCF2 locus on human chromosome 22, or into a cosYHZ304 genomic region onchromosome 2. When recombination between loxP sequences occurs by Cre,the promoter and GFP gene are joined together so that GFP is expressed.The recombinants can be sorted by FACS because they emit fluorescence.Since expression of GFP can be detected in a short time as describedabove, repetition of FACS sorting enables concentration of GFP positivecells faster than selection using a drug.

To increase the recombination frequency, it is assumed that the Crerecombination enzyme is expressed stably, not transiently. In this case,a chromosome, in which translocation occurs, may go back to the initialstate by re-recombination (or re-translocation) but with a lowfrequency, because translocation between two chromosomes occurs fromeach other. Hence, such an experiment generally needs a transientexpression of Cre recombination enzyme, or strict control over theexpression of Cre recombination enzyme. However, in this invention, atarget human artificial chromosome, in which translocation occurs, istransferred to a Chinese hamster CHO cell immediately aftertranslocation in a DT40 hybrid cell by the microcell-mediated chromosometransfer (herein after referred to as “MMCT.”), so that the targetchromosome in the CHO cell can avoid the effect of Cre recombinationenzyme. Therefore, no more strict expression control is required and Crerecombination enzyme can simply be expressed stably. As described below,the present invention is the first one that has succeeded intranslocation between two non-homologous human chromosomes in anon-human cell (a chicken DT-40 cell in this invention).

As disclosed in A, B, C and D above, the use of a series oftelomere-directed truncations and translocations between chromosomes bythe Cre-loxP system in a chicken DT-40 cell enables the cloning of humanchromosomal fragments with any size (exceeding the cloning size of YACvector) into loxP site (14p12) on stable SC20 chromosome vector. Thusthe inventors can announce a human artificial chromosome-constructingsystem. The human artificial chromosome-constructing system as disclosedherein is performed preferably using chicken DT-40 cells because thefrequency of homologous recombination is very low in normal culturedanimal cells. On the other hand, a mouse ES cell is known as anotheranimal cell with a high frequency of homologous recombination other thanchicken DT-40 cells (Shinichi Aizawa, Bio Manual Series 8, GeneTargeting, Yo-do sha, 1995). Thus mouse ES cells can also be used inconstructing human artificial chromosomes according to this invention.However, to culture mouse ES cells, which have an ability todifferentiate into various tissues, while maintaining them in anundifferentiated state, complex procedures like culture of vegetativecells, and other skills are required (Shinichi Aizawa, supra).Furthermore, mouse ES cells can differentiate depending on the types ofchromosomes to be introduced (WO97/07671). Accordingly, chicken DT-40cells are preferred for the construction of human artificial chromosomesbecause mouse ES cells have the problems as described above.

In addition to recombination between human chromosomes as illustrated inexamples, the translocation of a desired region (or a fragment) of ahuman chromosome to a mouse chromosome derived from a mouse ES cell isalso possible. This can be done by the procedures (1) to (4) as shownbelow.

(1) A recognition sequence, for example a loxP sequence, of asite-directed recombination enzyme is inserted by homologousrecombination into a chromosome of a mouse ES cell. A targeting vectorcomprising the loxP sequence contains a promoter sequence to express amarker, such as the following GFP. A preferable insertion site for theloxP sequence is, for example a subtelomere region of a mouse chromosomein order to avoid an effect on a gene of the mouse.(2) The following procedures A and B are performed in chicken DT-40cells which retain human chromosomes containing desired regions.(2)-A: A telomere sequence is inserted on the telomere side of a desiredregion on the human chromosome to shorten this chromosome. Thisprocedure may not be required when said desired region is located nearthe original telomere of said human chromosome.(2)-B: A recognition sequence, for example, a loxP sequence, is insertedon the centromere side of the desired region on the human chromosome. Atargeting vector comprising the loxP sequence comprises, for example aGFP gene.(3) The human chromosome (fragment) as produced in (2) above istransferred to a CHO cell by MMCT. Then the human chromosomal fragmentsare transferred from the CHO cell to the mouse ES cell prepared in (1)above.(4) Site-directed recombination enzyme is expressed in the mouse ES cellcomprising the human chromosome (or a fragment) as prepared in (3)above. In a cell wherein site-directed recombination occurs between theloxP sequences, the promoter sequence and GFP gene are joined together,and GFP is expressed. Thus these cells can be selected.

The above procedure enables mouse ES cells that have a chromosomecomprising a desired human chromosomal region (or a fragment) to beobtained through translocation. From the mouse ES cells, a chimericmouse or its progeny, stably retaining the human chromosomal region(fragment), can be produced.

The human artificial chromosome produced by the above human artificialchromosome-constructing system is preferably introduced into a Chinesehamster CHO cell before its introduction into mouse ES cells. WhenDieken et al. (Nature, Genet., 12:174, 1996) transferred modified humanchromosomes from chicken DT-40 cells to mouse MEL cells (a kind ofcancer cell), most were transferred in a fragmented state. When theinventors tried to transfer human chromosomes from chicken DT-40 cellsinto mouse A9 cells and the like, however, fragmentation was observedfor all the human chromosomes and no intact chromosomes could betransferred. However, upon transfer into Chinese hamster CHO cells, theinventors found for the first time that intact human chromosomes can betransferred. Therefore, the human artificial chromosomes prepared bythis human artificial chromosome-constructing system, are temporarilytransferred to CHO cells.

CHO cells are known to form microcells efficiently as mouse A9 cells do(for example, M. Koi et al., SCIENCE 260: 361, 1993). It should enableus to transfer human artificial chromosomes from CHO cells to mouse EScells and to produce chimeric mice retaining the human artificialchromosomes. Furthermore, it is known that SC20 fragments themselves canbe transferred to progeny. Thus it is naturally expected that humanartificial chromosomes (λ, κ-HAC) produced by this system using thesechromosomal fragments can be transferred to progeny.

In the afore-mentioned report of Tomizuka et al. (Nature Genet., 16:133, 199.7), the human chromosome introduced into a mouse was derivedfrom a human fibroblast. After introduction into a mouse ES cell via amouse A9 cell, it was functional in both a chimeric mouse and itsprogeny. Expression of the human gene on the introduced chromosome atthe transcription level and the protein level was confirmed for, forexample, a human antibody gene (Tomizuka et al., Nature Genet., 16: 133,1997). To date, it is still unknown if human chromosomes introduced intomouse cells via the cells of Aves, which is evolutionally distant frommammals, can keep their ability to function in mouse cells. Moreover,apart from being derived from birds, chicken DT-40 cells have a veryhigh homologous recombination efficiency (Takeda et al., Proc. Natl.Acad. Sci. USA, 89: 023, 1992). That is, a human gene on an introducedhuman chromosome may be inactivated by gene conversion between the humangene and homologous bird gene on a bird chromosome. Concerning thisproblem, there is only one report (Dieken et al., Nature Genet., 12:174, 1996) that when human chromosome 11 was transferred a mouse MELcell via a chicken DT-40 cell, transcripts of the human β-globin genewere detected. There is no report on the expression of a human gene atthe protein level and the function of expressed human proteins.

This invention shows that a human gene on a human chromosome transferredvia a chicken DT-40 cell, and a protein encoded by this gene arefunctional in a mouse. Human immunoglobulin λ chain protein was detectedin the sera of chimeric mice retaining human chromosome 22 fragment(comprising a human immunoglobulin λ chain gene) that had beengenetically engineered in chicken DT-40 cells. Furthermore, in responseto immunization with human serum albumin (HSA), increased antibody titerincluding antigen-specific human λ chain was confirmed. That is, theexpressed human immunoglobulin λ chain protein is present as aconstituent of a functional antibody molecule.

(2) Transfer of the Human Chromosome or Fragment thereof into a MousePluripotent Cell

It has been reported to date that an embyonic carcinoma cell (EC cell,Hanaoka et al., Differentiation, 48:83, 1991), an embryonic stem cell(ES cell, Evans, Nature, 292:154, 1981) or an embyonic germ cell (EGcell, Matsui et al., Cell, 70:841, 1992) that are derived from variousstrains of mice contribute to the normal somatic cells in mice, or arecapable of the production of chimeric mice, by injection into orcoculturing with a mouse early embryo. ES and EG cells have a very highability in this respect and in many cases, they also contribute to germcells thereby making it possible to produce progenies derived from thecells. EC cells can be obtained predominantly from teratocarcinoma; EScells from the inner cell masses of blastocysts; and EG cells fromprimordial germ cells appearing at the early stage of embryogeny. Thesecell lines and their mutants, and any undifferentiated cells that arecapable of differentiation into all or part of the normal somatic cellsin mice can be used as recipient cells for the transfer of humanchromosome in the present invention. In these recipient cells, for thepurpose of achieving advantageous expression of a human gene to beintroduced, a gene or genes such as a mouse gene homologous to the humangene can be disrupted in a chimeric mouse or a chimeric-mouse derivedtissue or cell by using homologous recombination in gene targeting(Joyner et al., Gene Targeting, 1993, IRL PRESS) or other techniques.

The microcells prepared from the human chromosome donor cells obtainedin item (1) or the microcells irradiated with γ-rays can be used asmaterials for the transfer of human chromosomes into the recipientcells. The human chromosome can be transferred into the recipient cellthrough fusion of the recipient cell with the microcell by the methoddescribed in Motoyuki Shimizu, “Cell Technology Handbook”, published byYodosha, 1992. The microcell donor cells retain markers by which humanchromosomes or fragments thereof can be selected in the recipient cells.The clone containing a gene, a chromosome or a fragment of interest canbe selected by PCR, Southern blot analysis, FISH method or the like inthe same manner as in (1), thus all kinds of human chromosomes orfragments thereof can be transferred. Moreover, if several chromosomesor fragments thereof which contain different selection markers aretransferred sequentially, a recipient cell retaining these chromosomesor fragments at the same time can be obtained. In addition, cloneshaving an increased number of the transferred chromosome can be selectedfrom the clones into which the human chromosome has been transferred.Such selection can be accomplished by increasing the concentration of aselection drug to be added to a culture medium.

In order to determine whether the recipient cell selected by the marker(e.g., G418 resistance) on the human chromosome retains the whole orpart of the chromosome retained by the donor cell, the followingconfirmative techniques may be employed: Southern blot analysis usingthe genomic DNA extracted from the selected recipient cell, with a humanspecific repeated sequence (L1, Alu, etc.: Korenberg et al., Cell,53:391, 1988) or a human gene used as a probe; and chromosome analysissuch as PCR method using a human gene specific primer or FISH methodusing a human chromosome specific probe (Lichter et al., Human Genetics,80:224, 1988).

(3) Production of a Chimeric Mouse from the Human Chromosome TransferredES Cell

The method described in Shinichi Aizawa, “Biotechnology Manual Series 8,Gene Targeting”, published by Yodosha, 1995 may be used to producechimeric mice from the ES cell clone obtained in (2). In selectingfactors for efficient production of chimeric mice, such as thedevelopmental stage of the host embryo and its strain, it is desired toemploy the conditions already reviewed for the respective ES cellclones. For example, 8-cell stage embryos derived from Balb/c (albino,CREA JAPAN, INC.) or ICR (albino, CREA JAPAN, INC.) are desirably usedfor CBAxC57BL/6 F1-derived TT2 cell (agouti, Yagi et al., AnalyticalBiochemistry, 214:70, 1993).

(4) Confirmation of the Retention of the Human Chromosome in theChimeric Mice and the Expression of a Human Gene

The contribution of the ES cells in mice produced from the embryos intowhich ES cells were injected can be roughly judged by the color of theircoat. However, it should be noted that the total absence of contributionto the coat color does not always lead to the conclusion that there isno contribution to other tissues. The detailed information on theretention of the human chromosome in various tissues of the chimericmice can be obtained by Southern blot analysis using the genomic DNAextracted from various tissues, by PCR or the like.

The expression of the gene on the transferred human chromosome can beconfirmed by the following methods. The expression of mRNA transcribedfrom the human chromosome can be detected by RT-PCR method or northernblotting (Ausubel et al., supra) using RNAs derived from various tissues(Kawasaki et al., P.N.A.S., 85:5698, 1988). The expression at theprotein level can be detected by enzyme immunoassay using an anti-humanprotein antibody that is rendered minimal in its ability to enter into across reaction with mouse homologous proteins (ELISA, Toyama and Ando,“Monoclonal Antibody Experiment Manual”, published by KodanshaScientific, 1987; Ishikawa, “Enzyme immunoassay with SuperhighSensitivity”, published by Gakkai Shuppan Center, 1993), westernblotting (Ausuel et al., supra), isozyme analysis utilizing thedifference in electrophoretic mobility (Koi et al., Jpn. J. Cancer Res.,80:413, 1989) or the like. The retention of the human chromosome in thechimeric mice and the expression of the gene on the human chromosome canbe confirmed by the appearance of the cells expressing a drug resistancemarker gene in primary culture cells derived from the chimeric mice.

For example, human IgM, IgG, IgA and the like in sera of the chimericmice which are produced from ES cells retaining human chromosome #14 onwhich a gene for human immunoglobulin heavy chain exists can be detectedby enzyme immunoassay using an anti-human Ig antibody that is renderedminimal in its ability to enter into cross reaction with mouse antibody.Hybridomas capable of producing a human immonoglobulin heavy chain canbe obtained by ELISA screening of hybridomas prepared by immunizing thechimeric mouse with a human-derived antigen (e.g., HSA) and fusing thespleen cells of the immunized mice with mouse myeloma cells (Toyama andAndo, “Monoclonal Antibody Experiment Manual”, published by KodanshaScientific, 1987).

The method for producing a chimeric non-human animal of the presentinvention has been explained above with reference to the case of a mouseretaining a human chromosome(s) or a fragment(s) thereof and expressingthe gene(s) on the chromosome(s) or fragment(s). In the presentinvention, chromosomes or fragments thereof to be transferred intochimeric non-human animals are not limited to those derived from humansbut include any foreign chromosomes and fragments thereof. The term“foreign chromosome” means a chromosome which is transferred into apluripotent cell and, subsequently, the gene on which (or a fragmentthereof) is expressed in a chimeric non-human animal. The organismspecies from which the foreign chromosome is derived is not particularlylimited. Other kinds of chimeric animals such as chimeric rat and pigcan be produced by the method of the present invention. ES cells orES-like cells derived from animals other than mouse were establishedwith rat (Iannaccone et al., Dev. Biol., 163, 288-, 1994), pig (Wheeleret al., Reprod. Fertil. Dev., 6, 563-, 1994) an bovine (Sims et al.,Proc. Natl. Acad. Sci. USA, 91, 6143-6147, 1994) and attempts have beenmade on cyprinodont, chicken and the like (“Transgenic Animal”,Protein•Nucleic Acid•Enzyme, October, 1995, Special Issue, published byKyoritsu Shuppan). It is known that sheep is developed normally from anunfertilized egg transplanted with the nucleus from ES-like cell (EDcell) or epithelial-like cell obtained by subcultivation of the ES-likecell through at least 10 generations (Campbell et al., Nature, 380, 64-,1996). These ES cells and ES-like cells can be used as recipient cellsin the transfer of foreign chromosomes to produce chimeric non-humananimals retaining the foreign chromosomes or fragments thereof andexpressing the genes on the chromosomes or fragments thereof in the samemanner as in the case of mouse.

Recently, rapid progression of nuclear transplantation technology allowsus to use the somatic cells from a fetus or adult, more differentiatedthan the above ES or ES-like cells, as donors for nucleartransplantation. There have been reports that viable progeny could beobtained by nuclear transplantation into unfertilized eggs using mammaryglandular cells of an adult sheep (Wilmut et al., Nature, 385: 810,1997), the fibroblasts of a fetal calf (Schnieke et al., Science,278:2130, 1997), or the cumulus cells of an adult mouse (Wakayama etal., Nature, 394: 369, 1998) as donors. Moreover, transgenic sheep(Schnieke et al., supra) and transgenic bovine (Cibelli et al., Science280: 1256, 1998) can also be generated using somatic cells, into whichcloned foreign DNA has been introduced, as donors.

Therefore, it is concluded that nuclear transplantation using somaticcells, into which foreign chromosomes or foreign recombinant chromosomeshave been introduced, as donors enables the generation of an animal thatretains and expresses the foreign chromosomes or the foreign recombinantchromosomes. This can be done, for example by the following procedures(1) to (5).

(1) The following steps A and B are performed in a chicken DT-40 cellthat retains human chromosome 22 comprising an Igλ region.(1)-A: A telomere sequence is inserted on the telomere side of the Igλregion on human chromosome 22 to shorten the chromosome.(1)-B: A recognition sequence of a site-directed recombination enzyme,for example a loxP sequence, is inserted on the centromere side (forexample HCF locus) of the Igλ region on human chromosome 22. A targetingvector comprising the loxP sequence comprises a promoter sequence forthe expression of a marker, such as a GFP gene as shown below.(2) A chicken DT-40 cell retaining human chromosome 14 fragment SC20 inwhich a loxP sequence and GFP gene are inserted on RNR2 locus, and thechicken DT-40 cell produced in (1) above is subjected to the whole cellfusion, thereby obtaining chicken DT-40 cells retaining two types ofhuman chromosomal fragments at the same time.(3) The site-directed recombination enzyme is expressed in a mouse EScell retaining the human chromosome (fragment) produced in (2) above.The promoter sequence and the GFP gene are joined together in the cell,wherein site-directed recombination occurs between the loxP sequences,so that the GFP is expressed. That is, chicken DT-40 cells comprisingrecombinant human chromosomes resulting from translocation can beselected.(4) The recombinant human chromosome (fragment) as produced in (3) aboveis transferred to a CHO cell by MMCT. A microcell is induced from theCHO cell retaining the obtained recombinant human chromosome (fragment),and then the microcell is fused with, for example a fibroblast derivedfrom a fetal calf. These fibroblasts, retaining the recombinant humanchromosomal fragments, can be selected by, for example the expression ofa drug-resistant marker, present on the recombinant human chromosome.(5) Nuclear transplantation using the fibroblast derived from a fetalcalf, retaining the recombinant human chromosomal fragments obtained in(4) above as a donor, and using a bovine unfertilized egg as arecipient, allows the generation of a bovine and offspring retaining andexpressing the recombinant human chromosomal fragments (Cibelli et al.,Science 280: 1256, 1998).

In the present invention, pluripotent cells into which a foreignchromosome(s) or a fragment(s) thereof are transferred are not limitedto the ES cells, EC cells and EG cells mentioned above. For example, itis possible to transfer a foreign chromosome(s) or a fragment(s) thereofinto bone marrow stem cells. If these bone marrow stem cells aretransplanted into a living organism, hereditary diseases, etc. may betreated.

If an ES cell retaining a foreign chromosome(s) or a fragment(s) thereofis differentiated to a germ cell in the chimeric non-human animal,reproduced progenies will retain the transferred chromosome(s) orfragment(s) thereof and express the gene(s) on the chromosome(s) orfragment(s) thereof.

The chimeric non-human animals or their progenies can be used to expressthe gene on the foreign chromosome or fragment thereof and to recoverthe expression product, thereby producing a biologically activesubstance. More specifically, the chimeric non-human animals or theirprogenies can be bred under the conditions for expressing the gene onthe foreign chromosome or fragment thereof to recover the expressionproduct from the blood, ascites and the like of the animals.Alternatively, the tissues or cells of the chimeric non-human animal, orimmortalized cells derived therefrom (e.g., hybridomas immortalized byfusion with myeloma cells) can be cultured under the conditions forexpressing the gene on the foreign chromosome or fragment thereof andthe expression product is thereafter recovered from the culture.Furthermore, a foreign chromosome(s) or a fragment(s) thereof which wasextracted from tissues or cells of these chimeric non-human animals ortheir progenies, or from immortalized cells derived therefrom; the DNAwhich is a component of said foreign chromosome(s) or fragment(s)thereof; or cDNA derived from the foreign chromosome(s) or fragment(s)thereof retained in tissues or cells of the chimeric non-human animalsor their progenies, or in immortalized cells derived therefrom may beused to transform animal cells or insect cells (e.g., CHO cells, BHKcells, hepatoma cells, myeloma cells, SF9 cells) and the transformedcells may be cultured under the conditions for expressing the gene onthe foreign chromosome(s) or fragment(s) thereof to recover theexpression product (e.g., an antibody protein specific to a particularantigen) from the culture. The expression product can be collected byknown techniques such as centrifugation and purified by known techniquessuch as ammonium sulfate fractionation, partition chromatography, gelfiltration chromatography, adsorption chromatography, preparativethin-layer chromatography and the like. The biologically activesubstance includes any kinds of substances encoded on foreignchromosomes, for example, antibodies, particularly human antibodies. Forexample, the human antibody gene on the foreign chromosome can be clonedfrom spleen cells of the chimeric non-human animal or immortalized cellssuch as hybridomas derived therefrom and transferred into Chinesehamster ovary cells (CHO), myeloma cells or the like to produce a humanantibody (Lynette et al., Biotechnology, 10:1121-, 1992; Bebbington etal., Biotechnology, 10:169-, 1992).

The chimeric mice or their progenies that retain human chromosomes #2,14 and/or 22 (or fragments thereof) which can be produced by the methodof the present invention can retain the greater part of the functionalsequences of respective genes for human antibody heavy chain onchromosome #14, light chain κ on chromosome #2 and light chain λ onchromosome #22. Hence, they can produce a wide repertory of antibodieswhich are more similar to human antibody repertory, compared with knowntransgenic mice into which parts of human antibody gene have beentransferred by using yeast artificial chromosomes and the like (Green etal., Nature Genetics, 7, 13-, 1994; Lonberg et al., Nature, 368, 856-,1994). Also, the chimeric mice and their progenies retaining two humanchromosomes (or fragments) of #2+#14, #22+#14 or other combination andthe mice and their progenies retaining three human chromosomes (orfragments) of #2+#14+#22 or other combination which are obtainable bymating said chimeric mice and their progenies retaining two humanchromosomes (or fragments), as produced by the method of the invention,can produce complete human antibodies both heavy- and light-chains ofwhich are derived from human. These mice can recognize human-derivedantigens as foreign substances to cause an immunoreaction with theantigens, thereby producing antigen-specific human antibodies. Theseproperties can be utilized to produce human monoclonal and polyclonalantibodies for therapeutic treatments (Green et al, supra; Longberg etal., supra). On the other hand, in order to obtain a human antibodyhaving high affinity for a particular antigen more efficiently, it isdesirable to produce a mouse which produces a human antibody but not amouse antibody (Green et al., supra; Lonberg et al., supra). In thepresent invention, this is achieved typically by the following Method Aor B using known techniques.

Method A: a method using a mouse antibody-deficient ES cell and a mouseantibody-deficient host embryo for chimera production.Method B: a method in which a progeny retaining a human chromosome isobtained from a human chromosome-transferred chimeric mouse, followed bymating said progeny with a mouse in a strain deficient in a mouseantibody gene.

A typical example for each of Methods A and B will be described belowspecifically.

Specific Procedures for Method A

1. One allele of a mouse antibody heavy-chain gene present in two copiesin a mouse ES cell is disrupted by homologous recombination in genetargeting (Joyner et al., “Gene Targeting”, published by IRL PRESS,1993). A marker gene, such as a G 418 resistance gene, sandwiched withtwo copies of a sequence which can be removed later by site-directedrecombination [for example, loxP sequence (see recombination with Crerecombinase in Sauer et al., supra; and see also the use of FLPrecombinase-FRT sequence in O'Gorman, Science, 251; 1351-, 1991)] isinserted at the site where the targeted gene is disrupted.2. The resultant drug-resistant mouse ES cells in which one allele of anantibody heavy-chain gene was disrupted is cultured in the presence ofthe drug at a high concentration. Then, those clones-which became highconcentration drug-resistant are selected. By screening these clones,clones in which both antibody heavy-chain genes were disrupted can beobtained (Shinichi Aizawa, supra).

Alternatively, the other allele of a target gene in the drug-resistantmouse ES cell in which one allele of the antibody heavy-chain gene hasbeen disrupted is also disrupted by homologous recombination. The sameprocedure may be repeated using a marker gene other than the precedinglyinserted marker gene. For example, homologous recombination is performedusing a G418-resistance gene, followed by another homologousrecombination using a puromycin-resistance gene to obtain clones inwhich both alleles of the antibody heavy-chain gene have been disrupted.When the same marker as the precedingly inserted marker is used, anenzyme gene that can cause site-directed recombination betweenrecombinant sequences inserted at the both ends of the drug-resistancegene of item 1 is transiently introduced. Subsequently, drug-sensitiveclones are selected that are free of the drug-resistance gene that hasbeen inserted in the target gene. Then, a marker gene is inserted againby homologous recombination in gene targeting to obtain clones in whichboth alleles of the target gene have been disrupted (Seishi Takatsu etal., Experimental Medicine, supplement, Basic Techniques forImmunological Study, p. 255-, 1995, Yodosha).

3. An enzyme gene (e.g., a Cre recombinase gene (Sauer et al., supra))which causes a site-directed recombination between the recombinationsequences inserted at both the ends of the drug-resistance gene in step1 above is transiently transferred into the mouse ES cells from step 2above in which both antibody heavy-chain genes were disrupted. Then,drug-sensitive clones are selected in which the drug-resistance genesinserted at the sites of both heavy-chain genes were deleted as a resultof recombination between the loxP sequences [Seiji Takatsu et al.,“Experimental Medicine (extra number): Basic Technologies inImmunological Researches”, p. 255-, published by Yodosha, 1995].4. The same procedures in steps 1-3 above are repeated for the mouseantibody light-chain κ gene to finally obtain drug-sensitive cloneswhich are completely deficient in antibody heavy-chain and light-chainκ.5. Human chromosome #14 (fragment) containing a human antibodyheavy-chain gene and marked with a drug-resistance gene (e.g., G418resistance gene) is transferred into the clone from step 4 above(antibody heavy-chain and light-chain κ-deficient mouse ES cell) bymicrocell fusion.6. Human chromosome #2 (fragment) or #22 (fragment) or both containing ahuman antibody light-chain gene(s) and marked with a drug-resistancegene different from the one used in step 5 above (e.g., puromycinresistance gene) are transferred into the clone obtained in step 5 aboveby microcell fusion.7. Chimeric mice are produced from the ES cells obtained in step 6 aboveby using embryos obtained from a mouse in a strain having no ability toproduce its own antibody (e.g., RAG-2 knockout mouse, Shinkai et al.,Cell, 68:855-, 1992; membrane-type μ chain knockout mouse, Kitamura etal., Nature, 350:423-, 1991) as host embryos.8. Most of the functional B lymphocytes in the resultant chimeric miceare derived from the ES cells [Seiji Takatsu et al., “ExperimentalMedicine (extra number): Basic Technologies in ImmunologicalResearches”, p. 234-, published by Yodosha, 1995]. Since those Blymphocytes are deficient in mouse heavy-chain and light-chain κ, theyproduce human antibodies alone mainly as a result of the expression ofthe functional human antibody genes on the transferred chromosomes.

Specific Procedures for Method B

1. Chimeric mice retaining a human chromosome or a fragment thereofcontaining human antibody heavy-chain, light-chain κ or light-chain λare used to produce a progeny which stably retains the human chromosomeor fragment thereof and which can transmit it to the next generation.2. A mouse in a strain which is homozygous regarding the deficiency inmouse antibody heavy-chain and light-chain κ and which retains humanchromosomes containing human antibody heavy-chain (#14)+light-chain κ(#2), heavy-chain (#14)+light-chain λ (#22) or heavy-chain(#14)+light-chain κ (#2)+light-chain λ (#22) is obtained by mating themouse in a strain expressing human antibody heavy-chain or light-chainfrom step 1 above or a mouse in a strain expressing both human antibodyheavy and light-chains obtained by mating the mice from step 1, with amouse in a strain deficient in its own antibody genes (e.g., themembrane-type μ chain knockout mouse mentioned above; light-chain κknockout mouse, Chen et al., EMBO J., 3:821-, 1993). Since mice in theresultant strain are deficient in mouse antibody heavy-chain andlight-chain κ genes, they produce human antibodies alone mainly as aresult of the expression of the functional human antibody genes on thetransferred chromosomes.

Both Method A and Method B may be used not only to yield humanantibodies but also to yield products of any genes located on a foreignchromosome efficiently.

The present invention will now be explained in greater detail withreference to the following examples, which do not limit the scope of thepresent invention.

EXAMPLE 1 Production of Chromosome Donor Cell Retaining Human Chromosome(Fragment) Labeled with G418 Resistance

Plasmid pSTneoB containing a G418 resistance gene (Katoh et al., CellStruct. Funct., 12:575, 1987; Japanese Collection of ResearchBiologicals (JCRB), Deposit Number: VE 039) was linearized withrestriction enzyme SalI (TAKARA SHUZO CO., LTD.) and introduced intohuman normal fibroblast cell HFL-1 (obtained from RIKEN Cell Bank,RCB0251). The HFL-1 cells were treated with trypsin and suspended inDulbecco's phosphate-buffered saline (PBS) at a concentration of 5×10⁶cells/ml, followed by electroporation using a Gene Pulser (Bio-RadLaboratories, Inc.) in the presence of 10 μg of DNA (Ishida et al.,“Cell Technology Experiment Procedure Manual”, published by Kodansha,1992). A voltage of 1000 V was applied at a capacitance of 25 μF with anElectroporation Cell of 4 mm in length (165-2088, Bio-Rad Laboratories,Inc.) at room temperature. The electroporated cells were inoculated intoan Eagle's F12 medium (hereinafter referred to as “F12”) supplementedwith 15% fetal bovine serum (FBS) in 3-6 tissue culture plastic plates(Corning) of 100 mmφ. After one day, the medium was replaced with a F12supplemented with 15% FBS and containing 200 μg/ml of G418 (GENENTICIN,Sigma). The colonies formed after 2-3 weeks were collected in 52 groupseach consisting of about 100 colonies. The colonies of each group wereinoculated again into a plate of 100 mmφ and cultured.

Mouse A9 cells (Oshimura, Environ. Health Perspect., 93:57, 1991; JCRB0211) were cultured in Dulbecco's modified Eagle's medium (hereinafterreferred to as “DMEM”) supplemented with 10% FBS in plates of 100 mmφ.The G418 resistant HFL-1 cells of 52 groups were cultured in F12supplemented with 15% FBS and 200 μg/ml of G418 in plates of 100 mmφ.The mouse A9 cells and HFL-1 cells were treated with trypsin and onefourth to one half of both cells were mixed. The mixed cells wereinoculated into a plate of 100 mmφ and cultured in a mixture of equalamounts of DMEM containing 10% FBS and F12 containing 15% FBS for aperiod ranging from a half day to one day. Cell fusion was carried outin accordance with the method described in Shimizu et al., “CellTechnology Handbook”, published by Yodosha, p. 127-, 1992. The cellsurface was washed twice with DMEM and then treated sequentially with 2ml of a PEG (1:1.4) solution for 1 minute and with 2 ml of PEG (1:3) for1 minute. After the PEG solution was sucked up, and the cells werewashed three times with a serum-free DMEM, followed by cultivation inDMEM supplemented with 10% FBS for 1 day. The cells were dispersed bytreatment with trypsin and suspended in a double selective medium (10%FBS supplemented DMEM) containing ouabain (1×10⁻⁵ M, Sigma) and G418(800 g/ml), followed by inoculation in 3 plates of 100 mmφ. After about3 weeks cultivation, the colonies formed were treated with trypsin todisperse the cells, which were cultured in a selective medium (10% FBSsupplemented DMEM) containing G418 (800 μg/ml)

The cells were dispersed by treatment with trypsin and two groups of thecells were collected, followed by cultivation in 6 centrifuge flasks(Coaster, 3025) of 25 cm² until the cell density reached 70-80%confluence. The medium was replaced with a medium (20% FBS supplementedDMEM) containing Colcemid (0.05 μg/ml, Demecolcine, Wako Pure ChemicalsCo., Ltd) and the cells were cultured for 2 days to form microcells.After the culture medium was removed, a cytochalasin B (10 μg/ml, Sigma)solution preliminarily warmed at 37° C. was filled in the 25 cm²centrifuge flask, which were inserted into an acryl centrifugecontainer, followed by centrifugation at 34° C. at 8,000 rpm for 1 hour.The microcells were suspended in a serum-free medium and purified bypassage through a filter. To the mouse A9 cells cultured to 80%confluence in the flask of 25 cm², the purified micorcells were addedand the two kinds of cells were fused with a PEG solution. The fusedcells were cultured in a G418 containing selective medium and coloniesformed were isolated. Human chromosomes #2, 4, 14 and 22 retained in therespective clones were identified by the methods described in (1)-(3)below. All other experimental conditions such as operating proceduresand reagents were in accordance with Shimizu et al., “Cell TechnologyHandbook”, published by Yodosha, p 127-.

(1) PCR Analysis

The isolated cells were cultured and genomic DNA was extracted from thecells with a Puregene DNA Isolation kit (Gentra System Co.) PCR wasperformed using the genomic DNA as a template with human chromosomespecific primers to select the clones retaining human chromosome #2, 4,14 or 22. The PCR amplification was conducted with about 0.1 μg of thegenomic DNA as a template, using a thermal cycler (GeneAmp 9600,Perkin-Elmer Corp.) in accordance with the method described in Innis etal., “PCR Experiment Manual”, published by HBJ Publication Office, 1991.Taq polymerase was purchased from Perkin-Elmer Corp. and the reactionwas performed in a cycle of 94° C., 5 minutes and 35 cycles ofdenaturing at 94° C., 15 seconds, annealing at 54-57° C., 15 seconds(variable with the primers) and extension at 72° C., 20 seconds. Thegene on each chromosome (O'Brien, Genetic Maps, 6th edition, Book 5,Cold Spring Harbor Laboratory Press, 1993) and polymorphic markers(Polymorphic STS Primer Pair, BIOS Laboratories, Inc.; Weissenbach etal., Nature 359:794, 1992; Walter et al., Nature Genetics, 7:22, 1994)were used as primers. The primers for the genes were prepared on thebasis of nucleotide sequences obtained from data bases such as GenBank,EMBL and the like. The names of the polymorphic primers and thesequences of the primers for the genes will be shown for the respectivechromosomes in the following examples (#2, Example 1; #4, Example 6,#14, Example 9; #22, Example 2). The following genetic markers andpolymorphic makers (Polymorphic STS Primer Pairs: D2S207, D2S177, D2S156and D2S159, BIOS Laboratories, Inc.) were used to identify chromosome#2.

C κ (immunoglobulin kappa constant): 5′-TGGAAGGTGGATAACGCCCT, (SEQ IDNO:1) TCATTCTCCTCCAACATTAGCA (SEQ ID NO:2) FABP1 (fatty acid bindingprotein-1 liver): 5′-GCAATCGGTCTGCCGGAAGA, (SEQ ID NO:3)5′-TTGGATCACTTTGGACCCAG (SEQ ID NO:4) Vk3-2 (imrnunoglobulin kappavariable): 5′-CTCTCCTGCAGGGCCAGTCA, (SEQ ID NO:5)5′-TGCTGATGGTGAGAGTGAACTC (SEQ ID NO:6) Vk1-2 (immunoglobulin kappavariable): 5′-AGTCAGGGCATTAGCAGTGC, (SEQ ID NO:7)5′-GCTGCTGATGGTGAGAGTGA (SEQ ID NO:8)

(2) Fluorescence In Situ Hybridization (FISH)

FISH analysis was conducted with probes specific to human chromosomes#2, 4, 14 and 22 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in accordancewith the method described in Matsubara et al., “FISH ExperimentProtocol”, published by Shujunsha, 1994.

For example, at least one clone retaining chromosome #2 was obtained in10 groups out of 26 groups (745 clones). Among them, only 5 clones werepositive to all the used primers specific to chromosome #2. FISHanalysis was conducted with these clones. FISH analysis was conductedwith probes specific to human chromosomes #2 (CHROMOSOME PAINTINGSYSTEM, Cambio Ltd.) in accordance with the method described inMatsubara et al., “FISH Experiment Protocol”, published by Shujunsha,1994. In the cells positive to all the primers, an intact form of humanchromosome #2 was observed. In some of the clones positive to part ofthe primers, an independent chromosome smaller than human chromosome #2was observed or a cell having a chromosome in a form fusing withchromosomes other than human chromosome #2 was observed (FIG. 1). InFIG. 1, the names of the clones are shown in the horizontal line and theprimers used in the PCR are shown in the left longitudinal line.  showspositive clones and X shows negative clones. The forms of humanchromosome #2 observed by FISH are shown in the bottom line. Nodescription means no performance of experiment.

A9 cells retaining human chromosomes #4, 14 and 22 were obtained by thesame procedure.

EXAMPLE 2 Transfer of Human Chromosome #22 into Mouse ES Cells byMicrocell Fusion

The mouse A9 cell clones retaining human chromosome #22 (hereinafterreferred to as “A9/#22”) from Example 1 were used as chromosome donorcells. Mouse ES cell line E14 (obtained from Martin L. Hooper; Hooper etal., Nature, 326:292, 1987) was used as a chromosome recipient cell.

E14 cells were cultured in accordance with the method described inAizawa Shinichi, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995 and G418 resistant STO cell line (obtained from Prof.Kondo Hisato, Osaka University) treated with mitomycin C (Sigma) wasused as a feeder cell. In the first step, microcells were prepared fromabout 10⁸ cells of A9/#22 in accordance with the method reported byShimizu et al. “Cell Technology Handbook”, published by Yodosha, 1992.The total amount of the resulting microcells were suspended in 5 ml ofDMEM. About 10⁷ cells of E14 were dispersed with trypsin and washedthree times with DMEM and suspended in 5 ml of DMEM. The cells were thenmixed with the microcells and the mixture was centrifuged at 1,250 rpmfor 10 minutes to remove the supernatant. The precipitate was dispersedby tapping and 0.5 ml of a PEG solution (1:1.4) [5g of PEG 1000 (WakoPure Chemicals Co., Ltd.) and 1 ml of DMSO (Sigma) as dissolved in 6 mlof DMEM] was added. The mixture was left to stand at room temperaturefor 1 minute and 30 seconds and 10 ml of DMEM was added slowly.Immediately thereafter, the resulting mixture was centrifuged at 1,250rpm for 10 minutes to remove the supernatant. The precipitate wassuspended in 30 ml of a medium for ES cells and inoculated into 3 tissueculture plastic plates (Corning) of 100 mm in diameter into which feedercells were inoculated. After 24 hours, the medium was replaced with amedium supplemented with 300 μg/ml of G418 (GENETICIN, Sigma) and mediumreplacements were thereafter conducted daily. Drug resistant coloniesappeared in 1 week to 10 days. The frequency of appearance was 0-5 per10⁷ of E14 cells. The colonies were picked up and grown. The cells weresuspended in a storage medium (a medium for ES cells+10% DMSO (Sigma))at a concentration of 5×10⁶ cells per ml and stored frozen at −80° C. Atthe same time, genomic DNA was prepared from 10⁶-10⁷ cells of each drugresistant clone with a Puregene DNA Isolation Kit (Gentra System Co.).

Human chromosome #22 was fragmented by irradiating the microcells with γrays (Koi et al., Science, 260:361, 1993). The microcells obtained fromabout 10⁸ cells of A9/#22 were suspended in 5 ml of DMEM and irradiatedwith γ rays of 60 Gy on ice with a Gammacell 40 (Canadian Atomic EnergyPublic Corporation) at 1.2 Gy/min for 50 minutes. The fusion of γray-irradiated microcells and the selection of drug resistant cloneswere conducted by the same procedure as in the case of the unirradiatedmicrocells. As a result, the frequency of the appearance of the drugresistant clones was 1-7 per 10⁷ of E14 cells. The drug resistant cloneswere stored frozen and DNA was prepared from the clones by the sameprocedure as in the case of the unirradiated microcells.

The retention of the transferred chromosomes in the unirradiatedmicrocell-transferred drug resistant clones E14/#22-9 and E14/#22-10,and in the γ ray-irradiated microcell-transferred drug resistant clonesE14/#22-14 and E14/#22-25 was confirmed by the methods described in(1)-(3) below.

(1) PCR analysis (FIG. 2)

The presence of the gene on human chromosome #22 (Genetic Maps, supra)and polymorphic markers (Polymorphic STS Primer Pairs: D22S315, D22S275,D22S278, D22S272 and D22S274, BIOS Laboratories, Inc.; Nature 359:794,1992) was detected by a PCR method using the genomic DNA of the drugresistant clone as a template. The sequences of oligonucleotide primersfor the genes prepared on the basis of nucleotide sequences obtainedfrom data bases such as GenBank, EMBL and the like are described below.

PVALB (parvalbumin):

PVALB (parvalbumin): 5′-TGGTGGCTGAAAGCTAAGAA, (SEQ ID NO:9)5′-CCAGAAGAATGGTGTCATTA (SEQ ID NO:10) MB (myoglobin):5′-TCCAGGTTCTGCAGAGCAAG, (SEQ ID NO:11) 5′-TGTAGTTGGAGGCCATGTCC (SEQ IDNO:12) DIA1 (cytochrome b-5 reductase): 5′-CCCCACCCATGATCCAGTAC, (SEQ IDNO:13) 5′-GCCCTCAGAAGACGAAGCAG (SEQ ID NO:14) Ig λ (immunoglobulinlambda): 5′-GAGAGTTGCAGAAGGGGTGACT, (SEQ ID NO:15)5′-GGAGACCACCAAACCCTCCAAA (sEQ ID NO:16) ARSA (arylsulfatase A):5′-GGCTATGGGGACCTGGGCTG, (SEQ ID NO:17) 5′-CAGAGACACAGGCACGTAGAAG (SEQID NO:18)

PCR amplification (Innis et al., supra) was conducted by using about 0.1μg of the genomic DNA as a template with the above 10 kinds of theprimers. As a result, amplification products having expected lengthswere detected when all the primers in the case of the two unirradiatedclones and with part of the primers in the case of the γ ray-irradiatedtwo clones. The results are shown in FIG. 2. In FIG. 2, a schematicchromosome map based on the G bands of human chromosome #22 and thelocation of some markers on bands are shown at the left side (O'Brien,GENETIC MAPS, 6th edition, BOOK 5, etc.). The arrangement of the geneticand polymorphic markers shows approximate positional relationships onthe basis of the presently available information (Science, HUMAN GENETICMAP, 1994; Nature Genetics, 7:22, 1994; Nature 359:794, 1992, etc.) andthe order is not necessarily correct. With respect to four kinds of theG418 resistant E14 cell clones, the markers for which the expectedamplification products were detected by PCR are shown by ▪ and themarkers for which the expected amplification products were not detectedare shown by □. The results of the observation by FISH analysis areshown at the bottom side. A9/#22 is a chromosome donor cell.

(2) Southern Blot Analysis

Southern blot analysis of about 2 μg of the genomic DNA digested withrestriction enzyme BglII (TAKARA SHUZO CO., LTD.) was conducted by usinghuman specific repeated sequence L1 (10⁴-10⁵ copies were present perhaploid genome, obtained from RIKEN DNA Bank; Nucleic acids research,13; 7813, 1985; pUK19A derived EcoRI-BamHI fragment of 1.4 kb) as aprobe in accordance with the method described in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., 1994. As aresult, a large number of bands hybridized with the human L1 sequencewere detected in DNA of each drug resistant clone. With respect to theunirradiated 2 clones, their patterns and the quantitative ratio ofhuman chromosomal DNA to mouse genomic DNA which could be presumed fromthe density of the respective bands were the same as those of A9/#22.The total signal intensity of the bands of the γ-ray irradiated clonescorrelated with the degree of the deletion confirmed by the PCRanalysis, as compared with that of A9/#22.

(3) Fluorescence In Situ Hybridization (FISH)

FISH analysis was conducted with probes specific to human chromosomes#22 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in accordance with themethod described in Matsubara et al., “FISH Experiment Protocol”,published by Shujunsha, 1994. As a result, in almost all of the observedmetaphase spreads, human chromosome #22 was detected in the form oftranslocation to the mouse chromosome with respect to E14/#22-9 and inthe form of an independent chromosome with respect to the three otherclones.

The results of the above experiments demonstrate that the obtained G418resistant clones E14/#22-9 and E14/#22-10 retained all or most part ofhuman chromosome #22 whereas the clones E14/#22-14 and E14/#22-25retained partial fragments of human chromosome #22.

EXAMPLE 3 Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #22

General procedures for obtaining mouse embryos, cultivation, injectionof the ES cells into the embryos, transplantation to the uteri of fostermothers were carried out in accordance with the method described inAizawa Shinichi, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995. The cells in a frozen stock of the G418 resistant ESclone E14/#22-9 which was confirmed to retain human chromosome #22 werethawed, started to culture and injected into blastcyst-stage embryosobtained by mating a C57BL/6XC3H F1 female mouse (CREA JAPAN, INC.) witha C3H male mouse (CREA JAPAN, INC.); the injection rate was 10-15 cellsper embryo. Two and half days after a foster mother [ICR or MCH(ICR)]mouse (CREA JAPAN, INC) was subjected to a pseudopregnant treatment,about ten of the ES cell-injected embryos were transplanted to each sideof the uterus of the foster mother. The results are shown in Table 1.

TABLE 1 Production of chimeric mice from the ES cells retaining humanchromosome #22 (fragments) Number of ES ES cell G418 cell-injectedNumber of Number of Contribution clone/human resistant blastocystoffspring chimeric to coat color chromosome clone No. stage embryos micemice <−10% 10-30% 30%< E14/#22 9 166 29 16 7 3 6

As a result of the transplantation of a total 166 of injected embryos,29 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of E14 cell-derived pale gray coat color in thehost embryo-derived agouti coat color (dark brown). Out of the 29offsprings, 16 mice were recognized to have partial pale gray coatcolor, indicating the contribution of the E14 cells. The maximumcontribution was about 40% in K22-22.

These results show that the mouse ES cell clone E14/#22-9 retaininghuman chromosome #22 maintains the ability to produce chimera, that is,the ability to differentiate into normal tissues of mouse.

EXAMPLE 4 Confirmation of Retention of Human Chromosomal DNA in VariousTissues of the Chimeric Mice Derived from the ES Cells Retaining HumanChromosome #22

In addition to the determination of coat color in Example 3, theretention of the transferred chromosome was confirmed by PCR analysisusing a template genomic DNA prepared from the tail of the chimericmouse. The tail was obtained from the chimeric mouse at least 3 weeksold in accordance with the method described in Motoya Katsuki,“Development Technology Experiment Manual”, published by KodanshaScientific, 1987. Genomic DNA was extracted from the tail with aPuregene DNA Isolation Kit. Out of the polymorphic primers used inExample 2, PVALB and D22S278 were used, with the extracted genomic DNAas a template, to confirm the amplification products. The analysis wasconducted with 10 of the mice in which the contribution to coat colorwas observed. As a result, the products of amplification with at leasteither of the primers were detected in all the mice.

Southern blot analysis was conducted in the same manner as in Example 2by using human L1 sequence as a probe with 2 μg of the genomic DNAderived from the tails of the 6 chimeric mice and one non-chimericmouse. As a result, the presence of a large number of human L1 sequencewas observed in all the chimeric mice and their patterns were similar tothose of E14/#22-9. The quantitative ratio to mouse genome was about 10%at maximum (FIG. 3). In FIG. 3, 2 μg of genomic DNA digested with BglIIwas used in each lane. Human L1 sequence labeled with ³²P was used as aprobe and signals were detected with Image Analyzer BAS2000 (Fuji PhotoFilm Co., Ltd.). The lanes represent the genomic DNA derived from thetails of the chimeric mice (K22-6, 7, 8, 9, 10, 11 and 12; 9 is thenon-chimeric mouse) and control DNA (C which is a mixture of E14/#22-9genomic DNA and E14 genomic DNA at a weight ratio of 1:9) as countedfrom the right. The DNA molecular weights are shown at the left side andchimerism in the chimeric mice at the right side (−: 0%, +: <10%, and++: 10-30%).

With respect to the chimeric mouse (K22-7) having about 5% contributionto coat color, genomic DNA was obtained from the brain, liver, muscle,heart, spleen, thymus, ovary and kidney with an ISOGEN (Nippon GeneCo.). For each tissue, PCR analysis was conducted with MB and D1A1selected from the primers for the genes used in Example 2. As a result,both primers gave expected amplification products in all the tissues.The results of PCR analysis using D1A1 primer are shown in FIG. 4. ThePCR products were electrophoresed on a 2% agarose gel and stained withethidium bromide for detection. The lanes in FIG. 4 represent thefollowing from the left: B. brain; L, liver; SM, skeletal muscle; H,heart; Sp, Spleen; Th, thymus; Ov, ovary; K, kidney; nc, non-chimericmouse tail-derived DNA (negative control); pc, human fibroblast cell(HFL-1) DNA (positive control).

These results show that E14/#22-9 contributed to various normal tissuesin the mouse and that it retained human chromosome #22.

EXAMPLE 5 Expression of the Human Genes in the Chimeric Mouse Derivedfrom the ES Cell Retaining Human Chromosome #22

The tail of the mouse (K22-7) having about 5% contribution to coat colorwas frozen with liquid nitrogen and then disrupted for use as a samplefor confirming the expression of the human genes. The sample was amixture of tissues such as skin, bones, muscles, blood and the like.Total RNA was extracted from the sample with an ISOGEN (Nippon Gene Co.)and used in an RT-PCR method to detect mRNAs of human myoglobin (MB) andhuman cytochrome b5 reductase (D1A1). The RT-PCR was performed inaccordance with the method described in Innis et al., “PCR ExperimentManual”, published by HBJ Publication Office, 1991. Randam hexameroligonucleotides (final concentration: 100 pmol, TAKARA SHUZO CO., LTD.)were used as primers for reverse transcription and Super Script (BRLCo.) as reverse transcriptase. The following primers were used foramplification using cDNA as a template.

MB: 5′-TTAAGGGTCACCCAGAGACT, (SEQ ID NO:19) 5′-TGTAGTTGGAGGCCATGTCC (SEQID NO:20) DIA1: 5′-CAAAAAGTCCAACCCTATCA, (SEQ ID NO:21)5′-GCCCTCAGAAGACGAAGCAG (SEQ ID NO:22)

As a result, amplification products specific to mRNAs of both genes weredetected (FIG. 5). The RT-PCR products were electrophoresed on a 2%agarose gel and stained with ethidium bromide for detection. In FIG. 5,M is a marker (HindIII digested λDNA+HaeIII digested φX174DNA, TAKARASHUZO CO., LTD.); MB, human myoglobin; D1A1, human cytochrome b5reductase; and WT, a wild-type C3H mouse.

With respect to the same individual (K22-7), total RNA was extractedfrom the brain, heart, thymus, liver, spleen, kidney, ovary and skeletalmuscle with an ISOGEN and RT-PCR was performed on each organ with theabove two primers. As a result, expected products of amplification withD1A1 were observed in all the organs and those with MB were observedonly in the heart and skeletal muscle (FIG. 6). Myoglobin is known to beexpressed specifically in muscle cells (Bassel-Duby et al., MCB,12:5024, 1992). Hence, the above results show that the gene on thetransferred human chromosome can be subjected to the normaltissue-specific regulation in the mouse. The PCR products wereelectrophoresed on a 2% agarose gel and stained with ethidium bromidefor detection. In FIG. 6, the lanes represent the following from theleft: B, brain; H, heart; Th, thymus; L, liver; Sp, spleen; K, kidney;ov, ovary; SM, skeletal muscle; and M, marker (supra). The lower bandobserved in the results of MB are believed to represent non-specificproducts.

These results show that the transferred human chromosome #22 canfunction in normal tissues of the chimeric mice.

EXAMPLE 6 Transfer of Human Chromosome #4 or Fragments Thereof into ESCells

The mouse A9 cell clone retaining human chromosome #4 (hereinafterreferred to as “A9/#4”) from Example 1 was used as a chromosome donorcell. Mouse ES cell line E14 (see Example 2) was used as a chromosomerecipient cell. The microcell fusion and the selection of G418 resistantclones were conducted by the same procedures as in Example 2. Thefrequency of the appearance of the drug resistant clones was 1-2 per 107of E14 cells. The drug resistant clones were stored frozen and genomicDNA were prepared by the same procedures as in Example 2. The retentionof the transferred human chromosome #4 or fragments thereof in the drugresistant clones E14/#4-4, E14/#4-7 and E14/#4-11 was confirmed by themethods described in (1)-(3) below.

(1) PCR Analysis (FIG. 7)

The presence of the gene on human chromosome #4 (O'Brien, Genetic Maps,6th edition, Book 5, Cold Spring Harbor Laboratory Press, 1993) andpolymorphic markers (Polymorphic STS Primer Pairs: D4S395, D4S412,D4S422, D4S413, D4S418, D4S426 and F11, BIOS Laboratories, Inc., Nature359:794, 1992) was detected by a PCR method. The sequences ofoligonucleotide primers for the genes prepared on the basis ofnucleotide sequences obtained from data bases such as GenBank, EMBL andthe like will be described below.

HD (huntington disease): 5′-TCGTTCCTGTCGAGGATGAA, (SEQ ID NO:23)5′-TCACTCCGAAGCTGCCTTTC (SEQ ID NO:24) IL-2 (interleukin-2):5′-ATGTACAGGATGCAACTCCTG, (SEQ ID NO:25) 5′-TCATCTGTAAATCCAGCAGT (SEQ IDNO:26) KIT (c-kit): 5′-GATCCCATCGCAGCTACCGC, (SEQ ID NO:27)5′-TTCGCCGAGTAGTCGCACGG (SEQ ID NO:28) FABP2 (fatty acid binding protein2, intestinal), 5′-GATGAACTAGTCCAGGTGAGTT, (SEQ ID NO:29)5′-CCTTTTGGCTTCTACTCCTTCA (SEQ ID NO:30)

PCR amplification was conducted with the above 11 kinds of the primers.As a result, the amplification products having expected lengths weredetected with all or part of the primers in all the three clones. In theE14/#4-4 and E14/#4-7 clones, the deletion of partial regions wasobserved. The results are shown in FIG. 7. In FIG. 7, a schematicchromosome map based on the G bands of human chromosome #4 and thelocation of some markers on bands are shown at the left side (seeExample 2). The arrangement of the genetic and polymorphic markers showsapproximate positional relationships on the basis of the presentlyavailable information (see Example 2) and the order is not necessarilycorrect. With respect to the three kinds of the G418 resistant E14 cellclones, the markers for which the expected amplification products weredetected are shown by ▪ and the markers for which the expectedamplification products were not detected are shown by □. The results ofthe observation by FISH analysis are shown at the lower side. A9/#4 is achromosome donor cell.

(2) Southern Blot Analysis (FIG. 8)

Southern blot analysis was conducted by the same procedure as in Example2 using human L1 sequence as a probe with genomic DNAs obtained fromE14/#4-4 and E14/#4-7. As a result, a large number of bands hybridizedwith the human L1 sequence were detected in DNAs of both drug resistantclones. The total signal intensity correlated with the degree of thedeletion confirmed by the PCR analysis, as compared with that of A9/#4.In FIG. 8, 2 μg of genomic DNA digested with BglII was used in eachlane. Human L1 sequence labeled with ³²P was used as a probe and thesignals were detected with an Image Analyzer (BAS 2000, Fuji Photo FilmCo., Ltd.). In FIG. 8, the lanes represent the following as counted fromthe left: 1, A9/#4 (chromosome donor cell); 2, A9/#4+A9 (1:2); 3,A9/#4+A9 (1:9); 4, A9; 5, E14/#4-7; and 6, E14/#4-4. Lanes 2 and 3represent mixtures of two kinds of DNAs at the ratios shown inparentheses. The molecular weights of DNAs are shown at the left side.

(3) Fluorescence In Situ Hybridization (FISH)

FISH analysis was conducted with probes specific to human chromosomes #4(CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) by the same procedure as inExample 2. As a result, in almost all of the observed metaphase spreadsof the three clones used, human chromosome #4 or partial fragmentsthereof were detected in the form of translocation to the mousechromosome with respect to E14/#4-4 and in the form of an independentchromosome with respect to the two other clones. The relative sizes ofthe observed human chromosome were consistent with those presumed fromthe results of the PCR analysis.

The results of the above experiments demonstrate that the obtained G418resistant clones retained the whole human chromosome #4 or partialfragments thereof.

EXAMPLE 7 Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #4 Fragments

The cells in frozen stocks of the G418 resistant ES cell clones E14/#4-4and E14/#4-7 which were confirmed to retain partial fragments of humanchromosome #4 were thawed, started to culture, and injected intoblastcyst stage embryos obtained by the same method as in Example 3; theinjection rate was 10-15 cells per embryo. Two and half days after afoster mother [ICR or MCH(ICR)] mouse (CREA JAPAN, INC.) was subjectedto a pseudopregnant treatment, about ten of the ES cell-injected embryoswere transplanted to each side of the uterus of the foster mother. Theresults are shown in Table 2.

TABLE 2 Production of chimeric mice from the E14 cell clones retaininghuman chromosome #4 (fragments) Number of ES ES cell G418 cell-injectedNumber of Number of Contribution clone/human resistant blastocystoffspring chimeric to coat color chromosome clone No. stage embryos micemice <10% 10-30% 30%< E14/#4 4 160 8 5 5 — — 7 80 5 2 1 1 —

As a result of the transplantation of a total of 240 injected embryos,13 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of E14 cell-derived pale gray coat color in thehost embryo-derived agouti coat color (dark brown). Out of the 13offsprings, 7 mice were recognized to have partial pale gray coat color,indicating the contribution of the E14 cells. The maximum contributionwas about 15% in one individual derived from E14/#4-7.

These results show that the mouse ES cell clones E14/#4-4 and E14/#4-7which retain fragments of human chromosome #4 maintain the ability toproduce chimera, that is, the ability to differentiate into normaltissues of mouse.

EXAMPLE 8 Confirmation of Retention of Human Chromosomal DNA in theChimeric Mice Derived from the ES Cells Retaining Partial Fragments ofHuman Chromosome #4 and Expression of the G418 Resistance Gene (1) PCRAnalysis

Using the chimeric mice produced in Example 7, genomic DNAs wereprepared from the tails of one individual derived from E14/#4-7(K#4-7-1: about 5% chimerism) and one individual derived from E14/#4-4(K#4-4-41: about 5% chimerism) by the same procedure as in Example 4.These DNAs were used as templates to conduct PCR analysis usingpolymorphic marker F11 for chromosome #4 analysis (see Example 6) whichwas detected in E14/#4-7 and E14/#4-4. As a result, expectedamplification products were detected in both mice.

(2) Southern Analysis (FIG. 9)

Southern analysis was conducted in the same manner as in Example 2 byusing human L1 sequence as a probe with 2 μg of the genomic DNA derivedfrom the tail of one individual derived from E14/#4-7 (K#4-7-1: about 5%chimerism). As a result, the presence of a large number of human L1sequence was observed and their patterns were similar to those ofE14/#4-7. The quantitative ratio to mouse genome was about 10% of thatof E14/#4-7 at maximum. In FIG. 9, 2 μg of genomic DNA digested withBglII was used in each lane. Human L1 sequence labeled with ³²P was usedas a probe and signals were detected with Image Analyzer BAS2000 (FujiPhoto Film Co., Ltd.). The molecular weights of DNAs are shown at theleft side. The lanes represent the following as counted from the left:1, K#4-7-1; 2, blank; and 3, E14/#4-7.

(3) Test on the Tail-Derived Fibroblast Cells for G418 Resistance

Fibroblast cells were prepared from the tails of one individual derivedfrom E14/#4-7 (K#4-7-1: about 5% chimerism) and one individual derivedfrom E14/#4-4 (K#4-4-41: about 5% chimerism). In the same procedure asin Example 4, the tail of each mouse was cut at a length of 5-10 mm andwashed several times with PBS/1 mM EDTA, followed by notching of thetail with a knife. The outer skin layer was removed and the innertissues were cut into fine pieces. The fine pieces of tissues weretransferred into a tube containing 5 ml of PBS/1 mM EDTA and left tostand for 30 minutes to 1 hour at room temperature. Subsequently, thesupernatant was removed leaving a 1 ml portion of the PBS/EDTA behind,and 1 ml of 0.25% trypsin/PBS was added. The tissues were dispersedthoroughly by tapping or pipetting at room temperature for 5-10 minutes.After centrifugation at 1,000 rpm for 10 minutes, the precipitate wassuspended in 2 ml of DMEM (10% FCS) and inoculated into a 35 mm plate.After cultivation for 7-10 days, the cells were treated with trypsin andabout 1 cells per plate were inoculated into two 35 mm plates. G418 wasadded to the medium in one plate at a final concentration of 400 μg/ml.The cells were cultured for 5-7 days and the appearance of viable cellsin each plate were examined. Under these conditions, 100% of thewild-type ICR mouse-derived fibroblast cells were killed in the presenceof G418. As a result, G418 resistant fibroblast cells was present inboth mice.

These results show that E14/#4-7 and E14/#4-4 contributed to variousnormal tissues in the mouse and that they retained partial fragments ofhuman chromosome #4.

EXAMPLE 9 Transfer of Human Chromosome #14 or Fragments Thereof intoMouse ES Cells

The mouse A9 cell clone retaining human chromosome #14 (hereinafterreferred to as “A9/#14”) from Example 1 was used as a chromosome donorcell. Mouse ES cell line TT2 (purchased from Lifetech Oriental Co., Yagiet al., Analytical Biochem., 214:70, 1993) was used as a chromosomerecipient cell. The TT2 cells were cultured in accordance with themethod described in Aizawa Shinichi, “Biomanual Series 8, GeneTargeting”, published by Yodosha, 1995 and G418 resistant primaryculture cells (purchased from Lifetech Oriental Co.) treated withmitomycin C (Sigma) were used as feeder cells. The microcell fusion andthe selection of G418 resistant clones were conducted by the sameprocedures as in Example 2. The frequency of the appearance of the drugresistant clones was 3-6 per 1 of TT2 cells. The drug resistant cloneswere stored frozen and genomic DNA was prepared by the same proceduresas in Example 2.

Human chromosome #14 was fragmented by irradiating the microcells withγ-rays (Koi et al., Science, 260:361, 1993). The microcells obtainedfrom about 10⁸ cells of A9/#14 were suspended in 5 ml of DMEM andirradiated with γ-rays of 30 Gy on ice with a Gammacell 40 (CanadianAtomic Energy Public Corporation) at 1.2 Gy/min for 25 minutes. Thefusion of γ ray-irradiated microcells and the selection of drugresistant clones were conducted by the same procedure as in the case ofthe unirradiated micorcells. As a result, the frequency of theappearance of the drug resistant clones was 3 per 1 of TT2 cells. Thedrug resistant clones were frozen stored and DNA was prepared by thesame procedure as in Example 2.

The retention of human chromosome #14 or partial fragments thereof inthe unirradiated microcell-transferred G418 resistant clones 1-4 and1-5, and in the G418 resistant clones 3-1 and 3-2 (a total of 4 clones)into which the γ-ray-irradiated microcell was transferred was confirmedby the methods described in (1) and (2) below.

(1) PCR Analysis (FIG. 10)

The presence of the gene on human chromosome #14 (O'Brien, Genetic Maps,6th edition, Book 5, Cold Spring Harbor Laboratory Press, 1993) andpolymorphic markers (Polymorphic STS Primer Pairs: D14S43, D14S51,D14S62, D14S65, D14S66, D14S67, D14S72, D14S75, D14S78, and PCI, BIOSLaboratories, Inc.; Nature 359:794, 1992; Nature Genetics, 7:22, 1994)was detected by a PCR method using genomic DNA of the drug resistantclone as a template. The sequences of oligonucleotide primers for thegenes prepared on the basis of nucleotide sequences obtained from databases such as GenBank, EMBL and the like are described below.

NP (nucleoside phosphorylase): 5′-ATAGAGGGTACCCACTCTGG, (SEQ ID NO:31)5′-AACCAGGTAGGTTGATATGG (SEQ ID NO:32) TCRA (T-cell receptor alpha):5′-AAGTTCCTGTGATGTCAAGC, (SEQ ID NO:33) 5′-TCATGAGCAGATTAAACCCG (SEQ IDNO:34) MYH6 (myosin heavy chain caraiac): 5′-TGTGAAGGAGGACCAGGTGT, (SEQID NO:35) 5′-TGTAGGGGTTGACAGTGACA (SEQ ID NO:36) IGA2 (immunoglobulinalpha-2 constant): 5′-CTGAGAGATGCCTCTGGTGC, (SEQ ID NO:37)5′-GGCGGTTAGTGGGGTCTTCA (SEQ ID NO:38) IGG1 (immunoglobulin gamma-1constant): 5′-GGTGTCGTGGAACTCAGGCG, (SEQ ID NO:39)5′-CTGGTGCAGGACGGTGAGGA (SEQ ID NO:40) IGM (immunoglobulin mu constant)5′-GCATCCTGACCGTGTCCGAA, (SEQ ID NO:41) 5′-GGGTCAGTAGCAGGTGCCAG (SEQ IDNO:42) IGVH3 (immunoglobulin heavy variable-3): 5′-AGTGAGATAAGCAGTGGATG,(SEQ ID NO:43) 5′-GTTGTGCTACTCCCATCACT (SEQ ID NO:44)

PCR amplification was conducted using the genomic DNAs of the 4 drugresistant clones as templates with the above 18 kinds of the primers bythe same procedure as in Example 2. As a result, expected amplificationproducts were detected with all or part of the primers. In the drugresistant clones 3-1 and 3-2 obtained by using the γ-ray irradiatedmicrocells, a tendency for the deletion of partial regions of chromosome#14 was observed. In the case where the unirradiated microcells wereused, deletion was observed as in the case of the 1-4 clone. The resultsare shown in FIG. 10. In FIG. 10, a schematic chromosome map based onthe G bands of human chromosome #14 and the location of some markers onbands are shown at the left side (see Example 2). The arrangement of thegenetic and polymorphic markers shows approximate positionalrelationships on the basis of the presently available information (seeExample 2) and the order is not necessarily correct. With respect tofour kinds of the G418 resistant TT2 cell clones, the markers for whichthe expected amplification products were detected are shown by ▪ and themarkers for which the expected amplification products were not detectedare shown by □. A9/#14 is a chromosome donor cell. The results ofExample 11 (1) are shown at the right side.

(2) Fluorescence In Situ Hybridization (FISH)

FISH analysis was conducted with probes specific to human chromosomes#14 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in accordance with themethod described in Matsubara et al., “FISH Experiment Protocol”,published by Shujunsha, 1994. As a result, in almost all of the observedmetaphase spreads of all the 4 clones, human chromosome #14 or partialfragments thereof were detected in the form of an independentchromosome. The relative sizes of the observed human chromosome wereconsistent with those presumed from the results of the PCR analysis.

The results of the above experiments demonstrate that the obtained G418resistant clones 1-4, 1-5, 3-1 and 3-2 retained the whole or partialfragments of human chromosome #14.

EXAMPLE 10 Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #14 or Fragments Thereof

The cells in the frozen stocks of four G418 resistant ES cell clones(1-4, 3-1, 3-2 and 1-5) that were prepared in Example 9 and which wereconfirmed to retain human chromosome #14 or fragments thereof werethawed, started to culture and injected into embryos at the eight-cellstage obtained by mating [ICR or MCH(ICR)] male and female mice (CREAJAPAN, INC.); the injection rate was 8-10 cells per embryo. The embryoswere cultured in an ES medium overnight to develop to blastocysts. Twoand half days after a foster mother ICR mouse (CREA JAPAN, INC.) wassubjected to a pseudopregnant treatment, about ten of the injectedembryos were transplanted to each side of the uterus of the fostermother. The results are shown in Table 3.

TABLE 3 Production of chimeric mice from the TT2 cell clones retaininghuman chromosome #14 (fragments) Number of ES ES cell G418 cell-injectedNumber of Number of Contribution clone/human resistant 8-cell offspringchimeric to coat color chromosome clone No. stage embryos mice mice <20%20-50% 50-80% TT2/#14 1-4 98 20 1 — — 1 1-5 110 14 2 1 — 1 3-1 103 11 21 1 — 3-2 183 19 3 — 2 1

As a result of the transplantation of a total of 494 injected embryos,64 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 64produced offsprings, 8 mice were recognized to have partial agouti coatcolor, indicating the contribution of the ES cells. The maximumcontribution was about 80% in one individual derived from 1-4.

These results show that the G418 resistant ES cell clones (1-4, 1-5, 3-1and 3-2) retaining human chromosome #14 or fragments thereof maintainthe ability to produce chimera, that is, the ability to differentiateinto normal tissues of mouse.

EXAMPLE 11 Confirmation of Retention of Human Chromosome #14 FragmentDNA in the Chimeric Mice Derived from the ES Cells Retaining HumanChromosome #14 Fragments

The retention of human chromosome #14 partial fragments in the chimericmice obtained in Example 10 was confirmed by the methods described in(1)-(3) below.

(1) PCR Analysis Using DNAS Derived from Various tissues

Genomic DNA was extracted from the tail of one individual derived from3-1 (K3-1-1: about 25% chimerism) by the same procedure as in Example 4.The DNA was used as a template to conduct PCR analysis using all of the14 primers for chromosome #14 analysis which were detected in 3-1. As aresult, expected amplification products were detected with all the 14primers (FIG. 10).

With respect to the same individual (K3-1-1), genomic DNA was obtainedfrom the brain, kidney, spleen, heart, liver and thymus with a PuregeneDNA Isolation Kit. For each tissue, PCR analysis was conducted with IGMprimers (see Example 9). As a result, expected amplification productswere detected in all the tissues (FIG. 11). The PCR products wereelectrophoresed on a 2% agarose gel and stained with ethidium bromidefor detection. In FIG. 11, the lanes represent the following as countedfrom the left: B, brain; K, kidney; Sp, Spleen; H, heart; L, liver; Th,thymus; pc, human fibroblast cell (HFL-1) DNA (positive control); nc,non-chimeric mouse tail DNA (negative control); and M, marker (HindIIIdigested λDNA+HaeIII digested φ X174 DNA, TAKARA SHUZO CO., LTD.).

(2) Test on the Tail-Derived Fibroblast Cells for G418 Resistance

Fibroblast cells were prepared from the tails of two individuals derivedfrom 3-2 (K3-2-1: about 25% chimerism, and K3-2-3: about 50% chimerism)and one individual derived from 1-4 (K1-4-1: about 80% chimerism). Inthe same procedure as in Example 4, the tail of each chimeric mouse of3-6 weeks was cut at a length of 5-10 mm and washed several times withPBS/1 mM EDTA, followed by notching of the tail with a knife. The outerlayer was removed and the inner tissues were cut into fine pieces. Thefine pieces of tissues were transferred into a tube containing 5 ml ofPBS/1 mM EDTA and left to stand for 30 minutes to 1 hour at roomtemperature. Subsequently, the supernatant was removed leaving a 1 mlportion of the PBS/EDTA behind, and 1 ml of 0.25% trypsin/PBS was added.The tissues were dispersed thoroughly by tapping or pipetting at roomtemperature for 5-10 minutes. After centrifugation at 1,000 rpm for 10minutes, the precipitate was suspended in 2 ml of DMEM (10% FCS) andinoculated into a 35 mm plate. After cultivation for 7-10 days, thecells were treated with trypsin and about 1 cells per plate wereinoculated into four 35 mm plates. G418 was added to the medium in twoof the plates at a final concentration of 400 μg/ml. The cells werecultured for 5-7 days and the viable cells in each plate were counted.Under these conditions, 100% of the wild-type ICR mouse-derivedfibroblast cells were killed in the presence of G418. Assuming the samegrowth rate of the G418 resistant fibroblast in the non-selective andselective media, the ratio of the viable cells in the selective mediumto those in the non-selective medium is believed to reflect thecontribution in the fibroblast cell populations of the G418 resistant EScell-derived fiblablast. As a result, the presence of G418 resistantfibroblast cells was observed in all the three individuals as shown inFIG. 12. In FIG. 12, % resistance is an average of 2 pairs of theselective/non-selective 35 mm plates for each mouse. ICR refers to thewild-type ICR mice.

(3) FISH Analysis of the Tail-Derived G418 Resistant Fibroblast Cells

FISH analysis of the K3-2-3 and K1-4-1 derived G418 resistant fibroblastcells obtained in (2) was conducted by the same procedure as in Example2. Total human DNA extracted from the HFL-1 cells (Example 1) waslabeled with FITC so that is could be used as a probe (Matsubara et al.,“FISH Experimental Protocol”, published by Shujunsha, 1994). As aresult, in almost all of the observed metaphase spreads of the bothindividuals, partial fragments of the human chromosome in independentforms were observed.

These results show that the TT2 cell clones retaining fragments of humanchromosome #14 contributed to various normal tissues in the mouseindividuals and that they retained partial fragments of human chromosome#14.

EXAMPLE 12 Transfer of Partial Fragments of Human Chromosome #2 into ESCells

The mouse A9 cell W23 retaining a human chromosome #2 fragment(hereinafter referred to as “A9/#2 W23”) from Example 1 was used as achromosome donor cell. Mouse ES cell line TT2 (see Example 9) was usedas a chromosome recipient cell. The microcell fusion and the selectionof G418 resistant clones were conducted by the same procedures as inExample 2. The frequency of the appearance of the drug resistant cloneswas 1-3 per 10⁷ of TT2 cells. The drug resistant clones were storedfrozen and genomic DNA was prepared by the same procedures as in Example2. The retention of partial fragments of human chromosome #2 in drugresistant clones 5-1, 5-2 and 5-3 was confirmed by the methods describedin (1) and (2) below.

(1) PCR Analysis

The presence of Cκ and FABP1 that are the genes on human chromosome #2(Genetic Maps, supra) and which were detected in the chromosome donorcell A9/#2 W23 was detected by a PCR method.

As a result of PCR amplification using each primer, expectedamplification products were detected with both primers in all of the 3clones.

(2) Fluorescence In Situ Hybridization (FISH)

FISH analysis was conducted with probes specific to human chromosome #2(CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) by the same method as inExample 2. As a result, in almost all of the observed metaphase spreadsof the 3 clones, partial fragments of human chromosome #2 in the form ofindependent chromosomes were detected. The sizes of the observed humanchromosome were the same as those observed in A9/#2 W23.

The results of the above experiments demonstrate that the obtained G418resistant clones retained partial fragments of human chromosome #2.

EXAMPLE 13 Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #2

The cells in a frozen stock of the G418 resistant ES cell clone 5-1 thatwas obtained in Example 12 and which was confirmed to retain humanchromosome #2 was thawed, started to culture and injected into 8-cellstage embryos obtained by mating ICR or MCH(ICR) male and female mice(CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo. Theembryos were cultured in an ES medium (Example 9) overnight to developto blastocysts. Two and half days after a foster mother ICR mouse (CREAJAPAN, INC.) was subjected to a pseudopregnant treatment, about ten ofthe injected embryos were transplanted to each side of the uterus of thefoster mother. The results are shown in Table 4.

TABLE 4 Production of chimeric mice from the TT2 cell clone retaininghuman chromosome #2 (fragments) Number of ES ES cell G418 cell-injectedNumber of Number of Contribution clone/human resistant 8 cell offspringchimeric to coat color chromosome clone No. stage embryos mice mice <20%20-50% 50-80% TT2/#2 5-1 264 51 18 7 5 6 (W23)

As a result of the transplantation of a total of 264 injected embryos,51 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 51produced offsprings, 18 mice were recognized to have partial agouti coatcolor, indicating the contribution of the ES cells. The maximumcontribution was about 80%.

These results show that the G418 resistant ES cell clone (5-1) retaininga fragment of human chromosome #2 maintains the ability to producechimera, that is, the ability to differentiate into normal tissues ofmouse individual.

EXAMPLE 14 Detection of Human Antibody Heavy Chain in Sera of the HumanChromosome #14 Transferred Chimeric Mice

The concentrations of human antibody in the sera were determined byenzyme-linked immunosorbent assay (ELISA). The ELISA for human antibodywas performed in accordance with the method described in Toyama andAndo, “Monoclonal Antibody Experiment Manual”, published by Kodansha,1987; Andou and Chiba, “Monoclonal Antibody Experiment ProcedureManual”, published by Kodansha Scientific, 1991; Ishikawa, “Super HighSensitivity Enzyme Immuno Assay”, published by Gakkai-syuppan center,1993; Ed Harlow and David Lane, “Antibodies A Laboratory Manual”,published by Cold Spring Harbor Laboratory, 1988 and A. Doyle and J. B.Griffiths, “Cell & Tissue Culture: Laboratory Procedures”, published byJohn Wiley & Sons Ltd., 1996. In some assays, the condition of reactionwere modified, for example, the reaction was performed at 4° C. overnight. Antibodies to human-immunogloblin or antigen were diluted toabout 0.5-10 μg/ml (100-5000 fold) and ELISA plates were coated withthese solutions. PBS supplemented with 5% mouse serum (Sigma, M5905) wasused for blocking and dilution of the samples and labeled antibodies.PBS was used for 20-fold dilution of the chimeric mouse sera. Afterwashed, the coated plate was blocked over 1 hour. After plate waswashed, sample was added and incubated over a half hour. After washed,Enzyme labeled anti-human immunogloblin antibodies diluted 100-5000folds were added to the plates and incubated over 1 hour, the plate waswashed and then substrate was added. In some assays, the same procedurewas applied except that a biotin-labeled antibody was used. After platewas washed, avidin-enzyme complex was added. After plate was washed,substrate was added. Absorbances were measured with a microplate reader(Bio-tek instrument, EL312e). The chimeric mice (Example 10, K3-1-2,K3-2-2 and K3-2-3) which were 29-35 days old were bled and assayed byELISA. Anti-human IgM mouse monoclonal antibody (Sigma, 16385) wasdiluted with 50 mM carbonate-bicartonate buffer (pH 9.6) and absorbed tothe 96-well microtiter plates. The serum samples diluted with mouseserum (Sigma, M5905) supplemented PBS were added to the plates.Subsequently, peroxidase-labeled anti-human IgM goat antibody (Tago,2392) was added and the plates were incubated. After ABTS substrate(Kirkegaard & Perry Laboratories Inc., 506200) was added, enzymeactivity was determined by absorbance measurement at 405 nm. Purifiedhuman IgM antibody (CAPEL, 6001-1590) and IgG (Sigma, 14506) were usedas standards. The standards were diluted stepwise with mouseserum-supplemented PBS. In the determination of human IgG concentration,anti-human IgG goat antibody (Sigma, 13382) was absorbed to the plateand the human IgG was detected with peroxidase-labeled anti-human IgGgoat antibody (Sigma, A0170). The results are shown in Table 5. Bothhuman IgM and IgG were detected.

TABLE 5 Concentrations of Human Antibodies in Chimeric Mouse Sera(ELISA) Chimeric Mouse (mg/l) IgG (mg/l) IgM K3-1-2 0.37 3.7 K3-2-2 0.335.9 K3-2-3 0.51 3.4

Two milliliters of human serum albumin (HSA, Sigma, A3782) dissolved inPBS was mixed with adjuvant (MPL+TDM Emulsion, RIBI Immunochem ResearchInc.) to prepare an antigen solution at a concentration of 0.25 mg/ml.The chimeric mice retaining human chromosome #14 fragment (Example 10,K3-1-1 and K3-2-1) were immunized with 0.2 ml of the antigen solution 3times at days 27, 34 and 41 after birth. The chimeric mouse sera wereassayed by ELISA. The results are shown in FIGS. 13 and 14. The humanantibody concentration in the sera of the HSA-immunized chimeric micewas increased after the immunization. In the K3-1-1 mouse, 18 μg/ml ofhuman IgM and 2.6 μg/ml of IgG were detected in the serum at day 17after the immunization. In the serum of the control ICR mouse, the humanantibody titer was not significant.

EXAMPLE 15 Production of Human Antibody Heavy Chain-Producing Hybridomasfrom the Human Chromosome #14 Transferred Chimeric Mouse

The spleen was removed from the human albumin-immunized chimeric mouse(K3-1-1, Example 14) at day 44 after birth. The spleen cell was fusedwith a myeloma cell to produce a hybridoma. The hybridoma was producedusing a myeloma cell P3X63Ag8.653 (DAINIPPON PHARMACEUTICAL CO., LTD.,05-565) by the method described in Ando and Chiba, “Monoclonal AntibodyExperimental Procedure Manual”, published by Kodansha Scientific, 1991.The hybridomas were inoculated into ten 96-well plates and cultured for1 week. The culture supernatant was analyzed by ELISA. The ELISAprocedure was conducted by using anti-human IgM mouse monoclonalantibody (Sigma, I6385) immobilized on ELISA plate in the same manner asin Example 14 to give 6 positive clones. HSA (antigen) was dissolved in50 mM carbonate-bicarbonate buffer (pH 9.6) at a concentration of 5μg/ml and the antigen solution was dispensed in 100 μl portions into allthe wells of the ELISA plates. After the addition of the supernatant,peroxidase-labeled anti-human IgA+IgG+IgM goat antibodies (Kierkegaard &Perry Laboratories Inc., 04-10-17) were used for detection ofHSA-specific human antibody. One positive clone was confirmed in the tenplates. This clone was one of the 6 human IgM positive clones. The clone(H4B7) was further cultured and the culture supernatant was diluted,followed by ELISA analysis using HSA as an antigen withperoxidase-labeled anti-human IgM goat antibody (Tago, 2392) in the samemanner as described above. As a result, the absorbance decreased withthe increase in the dilution of the culture solution. Serial twofolddilutions of 2 μg/ml human IgM (CAPEL, 6001-1590) showed low absorbanceregardless of dilution ratios. This suggests that the antibody producedby hybridoma H4B7 had a specificity to HSA (FIG. 15). In FIG. 15, thedilution of the culture supernatant samples is plotted on the horizontalaxis and the absorbance at 405 nm is plotted on the vertical axis.

EXAMPLE 16 Re-Marking of the G418 Resistance-Marked Human Chromosome #2Fragment with Puromycin Resistance

The A9 cells retaining the G418 resistance-marked human chromosome #2fragment (W23) (see Example 1, FIG. 1) were cultured in a G418 (800μg/ml) containing selective medium (10% FBS, DMEM) in a 100 mm plate.

Plasmid pPGKPuro (provided by Dr. Peter W. Laird (WHITEHEAD INSTITUTE))containing puromycin resistance gene was linearized with restrictionenzyme SalI (TAKARA SHUZO CO., LTD.) before transfection. The cells weretreated with trypsin and suspended in Dulbecco's phosphate bufferedsaline (PBS) at a concentration of 5×10⁶ cells/ml, followed byelectroporation using a Gene Pulser (Bio-Rad Laboratories, Inc.) in thepresence of 10 μg of DNA in the same manner as in Example 1. A voltageof 1000 V was applied at a capacitance of 25 μF with an ElectroporationCell of 4 mm in length (Example 1) at room temperature. Theelectroporated cells were inoculated into media in 3-6 plates of 100 mmφAfter one day, the medium was replaced with a double-selective mediumcontaining 10 μg/ml of puromycin (Sigma, P-7255) and 800 μg/ml of G418.The colonies formed after 2-3 weeks were collected in groups eachconsisting of about 200 colonies. The cells of each of the three groupswere cultured in two or three 25 cm² flasks to form microcells. Themouse A9 cells were cultured in a 25 cm² flask and fused with themicrocells by the same procedure as in Example 1. The fused cells weretransferred into two 100 mm plates and cultured in the double-selectivemedium containing G418 and puromycin. One of the three groups gave twodouble-drug resistant clones. In these clones, it was most likely thatpuromycin resistance marker had been introduced into human chromosome #2fragment.

EXAMPLE 17 Duplication of Transferred Human Chromosome in the HumanChromosome Transferred ES Cells

The ES cell clone retaining the G418 resistance marked human chromosome#14 fragment (E14/#14-36) was cultured in a medium containing G418 at ahigh concentration to give ES cell clones in which the human chromosomewas duplicated (“Biomanual Series 8, Gene Targeting”, published byYodosha, 1995). G418 resistant mouse primary cells (purchased fromLifetech Oriental) were inoculated into a 100 mm plate without treatingwith mitomycin C and used as feeder cells. The E14/#14-36 cells wereinoculated into the 100 mm plate and after half a day, the medium wasreplaced with a medium containing G418 at a concentration of 16 mg/ml.The medium was replaced every 1-2 days. The G418 concentration waschanged to 10 mg/ml one week later and the cultivation was continued.Among the colonies formed, 15 were picked up and cultured, followed byFISH analysis of chromosome using human chromosome #14 specific probes(see Example 9). As a result, human chromosome #14 fragment was found tohave duplicated in the 8 clones.

EXAMPLE 18 Preparation of Mouse ES Cells Retaining Both Human Chromosome#2 Partial Fragments and Human Chromosome #14 Partial Fragments

In a microcell transfer experiment using the double-drug resistant clonePG-1 from Example 16 as a microcell donor cell and a wild-type A9 cellas a recipient cell, it was confirmed that the human chromosome #2partial fragment retained in PG-1 was marked with a puromycin resistancegene. The preparation of microcells and the fusion with the A9 cells wascarried out by the same methods as in Example 1. As a result, 10 daysafter the microcell fusion, a total of fifty nine G418 resistantcolonies appeared. After the medium for these colonies was changed toone containing 8 μg/ml puromycin, the colonies were cultured for 3 daysto give 45 viable colonies (76%). In many cases of microcell fusion,only one or few chromosomes are transferred into a recipient cell.Hence, cotransfer of both the resistance genes at a high frequency showsthat the G418 resistance-labeled chromosome #2 partial fragment retainedin the PG1 clone was also marked with the puromycin resistance gene. Inaddition, for the detection of the respective marker genes on the humanchromosome #2 partial fragment, FISH analysis was conducted by usingpSTneoB (see Example 1) as a probe in the case of the A9/#2 W23 clonehaving only G418 resistance (see Example 16) and by using pPGKPuro (seeExample 16) as a probe in the case of the PG1 clone in accordance withthe method described in Matsubara et al., “FISH Experiment Protocol”,published by Shujunsha, 1994. As a result, in the case of the A9/#2 W23clone, one signal was observed in each of the sister chromatids of thehuman chromosome #2 partial fragment observed in Example 12 (2 signalsin total). This indicated the insertion of pSTneoB into the humanchromosome #2 partial fragment at one site. In the case of the PG1clone, a total of 4 signals were observed on a chromosome fragment ofthe same size as in A9/#2 W23. Since pSTneoB and pPGKPuro had identicalsequences in their vector portions, the pSTneoB could be detected by thepPGKPuro probe. Hence, it is believed that out of the four signalsobserved in the PG1 clone, two were from the pSTneoB and the other twowere from the pPGKPuro. These results show that the human chromosome #2partial fragment retained in the PG1 was marked with both the G418 andpuromycin resistances.

The PG1 cell clone was used as a chromosome donor cell to prepare amouse ES cell retaining both a human chromosome #2 partial fragment anda human chromosome #14 partial fragment. The G418 resistant TT2 cellclone 1-4 already retaining the human chromosome #14 partial fragment(see Example 9) was used as a chromosome recipient cell. The microcellfusion and the selection of puromycin resistant cells were carried outby the same methods as in the selection of the G418 resistant clones inExample 9 except that the concentration of puromycin was 0.75 μg/ml. Thefrequency of the appearance of the resulting puromycin resistant cloneswas 3-7 per 107 of 1-4 cells. The presence of G418 resistance in thesepuromycin resistant clones was confirmed from the fact that they weregrown in the presence of 300 μg/ml of G418. The double-drug resistantclones were stored frozen and genomic DNA was prepared by the samemethods as in Example 2. The retention of the human chromosome #2partial fragment and human chromosome #14 partial fragment was confirmedby the method described in (1) in the case of double-drug resistantclones PG5, PG15 and PG16 and by the method described in (2) in the caseof the clone PG15.

(1) PCR Analysis

Genomic DNAs of the double-drug resistant clones were used as templatesin the PCR amplifications. Among the markers on human chromosomes #2 and#14 (Genetic Maps, supra), the primers whose presence in the A9/#2 W23clone was confirmed in Example 12 and those whose presence in theTT2/#14 1-4 clone was confirmed in Example 9 were used. All the primersgave expected amplification products in all the three clones.

(2) Fluorescence In Situ Hybridization (FISH)

FISH analysis was conducted by using FITC-labeled human total DNA as aprobe in the same manner as in Example 11. As a result, in almost all ofthe metaphase spreads, two (large and small) human chromosome fragmentswere detected. The large fragment had the same size as that of thepartial fragment detected by using the human chromosome #14 specificprobes in the case of the TT2/#14 1-4 clone in Example 9 and the smallfragment had the same size as that of the partial fragment detected byusing the human chromosome #2 specific probes in the case of the TT2/#25-1 in Example 12. The results are shown in FIG. 16. In FIG. 16, theless bright chromosome was derived from the mouse. The two (large andsmall) chromosome fragments of high brightness due to FITC fluorescenceas shown by arrows were derived from the human, which are believed tocorrespond to the human chromosome #14 and #2 partial fragments.

These results show that the obtained double-drug resistant ES clonesretained both the human chromosome #2 partial fragment and the humanchromosome #14 partial fragment.

EXAMPLE 19 Production of Chimeric Mice from the Mouse ES Cell ClonesRetaining Both Human Chromosome #2 Partial Fragments and HumanChromosome #14 Partial Fragments

The cells in frozen stocks of the G418 and puromycin double-resistantTT2 cell clones PG5, PG15 and PG16 from Example 18 which were confirmedto retain human chromosome #2 partial fragments and human chromosome #14partial fragments were thawed, started to culture and injected into8-cell stage embryos obtained by mating ICR or MCH(ICR) male and femalemice (CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo.The embryos were cultured in a medium for ES cells (see Example 9)overnight to develop to blastocysts. Two and a half day after a fostermother ICR mouse was subjected to a pseudopregnant treatment, about tenof the injected embryos were transplanted to each side of the uterus ofthe foster mother. The results are shown in Table 6.

TABLE 6 Production of chimeric mice from the mouse ES cell clonesretaining both human chromosome #2 partial fragments and humanchromosome #14 partial fragments Number of ES ES cell Double-drugcell-injected Number of Number of Contribution clone/human resistant8-cell stage offspring chimeric to coat color chromosome clone No.embryos mice mice <10% 10-50% 50%< TT2/ PG5  160 26 8 7 1 — #14 + #2PG15 168 15 3 1 2 — PG16 223 32 12 3 6 3

As a result of the transplantation of a total of 551 injected embryos,73 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 73produced offsprings, 23 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cells.

These results show that the ES cell clones PG5, PG15 and PG16 retaininghuman chromosome #2 partial fragments and human chromosome #14 partialfragments maintain the ability to produce chimera, that is, the abilityto differentiate into normal tissues of mouse.

EXAMPLE 20 Detection of Human Antibody in Sera of the Chimeric MiceDerived from the ES Cells Retaining Both Human Chromosome #2 PartialFragments and Human Chromosome #14 Partial Fragments

The two KPG-15 (9 weeks old; derived from the PG-5 clone, 10% chimerism)and KPG-18 (5 weeks old; derived from the PG-5 clone, 10% chimerism)chimeric mice from Example 19 were immunized with 0.2 ml of a solutionof human serum albumin (HSA, Sigma, A3782) and adjuvant (MPL+TDMEmulsion, RIBI Immunochem Research Inc.) at a HSA concentration of 0.25mg/ml. The chimeric mice were bled just before the immunization and 8days after that and the concentrations of human antibody μ and κ chainsin the sera were determined by ELISA (see Example 14). Ninety six-wellmicrotiter plates were coated with anti-human antibody κ chain goatantibody (VECTOR LABORATORIES INC., AI-3060) diluted with 50 mMcarbonate-bicarbonate buffer (pH 9.6) and then a serum sample dilutedwith mouse serum (Sigma, M5905)-containing PBS was added. Subsequently,biotin-labeled anti-human antibody κ chain goat antibody (VECTORLABORATORIES INC., BA-3060) was added to the plates and incubated. Acomplex of biotinylated horseradish peroxidase and avidin DH (VECTORLABORATORIES, INC., Vectastain ABC Kit, PK4000) was added and incubated.After 3,3′,5,5′-tetramethylbenzidine (TMBZ, Sumitomo Bakelite, ML-1120T)was added as a peroxidase substrate, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG antibody having κchain (Sigma, I-3889) was used as standard. The standard was dilutedstepwise with mouse serum-supplemented PBS. In the case of μ chain,96-well microtiter plates were coated with anti-human antibody μ chainmouse monoclonal antibody (Sigma, I-6385) diluted with 50 mMcarbonate-bicarbonate buffer (pH 9.6) and then a serum sample was added.Subsequently, peroxidase-labeled anti-human antibody μ chain mouseantibody (The Binding Site Limited, MP008) was added to the plates andincubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzymeactivity was determined by absorbance measurement at 450 nm. Purifiedhuman IgM antibody having μ chain (CAPPEL, 6001-1590) was used asstandard. The standard was diluted stepwise with mouseserum-supplemented PBS. As a result, both the human antibody μ and κchains were detected in both individuals. The concentrations of thesehuman antibodies in the sera increased after the immunization (Tables 7and 8).

TABLE 7 Concentrations of Human Antibodies in Chimeric Mouse KPG15(ELISA) IgM (mg/l) Igκ (mg/l) Before Immunization 0.19 1.6 8 Days AfterImmunization 0.75 1.7

TABLE 8 Concentrations of Human Antibodies in Chimeric Mouse KPG18(ELISA) IgM (mg/l) Igκ (mg/l) Before Immunization 0.29 0.57 8 Days AfterImmunization 3.4 0.87

These results show that human antibody heavy and light chain genes canfunction in the chimeric mice derived from the ES cells retaining bothhuman chromosome #2 partial fragments and human chromosome partialfragments.

EXAMPLE 21 Detection of Anti-HSA Human Antibody 7 Chain in Sera of theHuman Chromosome #14 Fragments Transferred Chimeric Mice

The chimeric mice retaining human chromosome #14 fragments which wereproduced by the same method as in Example 10 (K9 and K11: both werederived from the TT2 cell clone 3-2, with chimerisms of 50% and 30%,respectively) were immunized with HSA either 4 times at days 79, 93, 107and 133 after birth (K9) or 3 times at days 74, 88 and 111 after birth(K11) by the same method as in Example 20. Antibodies including human 7chain against human serum albumin in the sera of the chimeric mice weredetected by ELISA. Ninety six-well microtiter plates were coated withHSA (Sigma, A 3782) diluted with 50 mM carbonate-bicarbonate buffer (pH9.6) and then a sample diluted with PBS was added. Subsequently,peroxidase-labeled anti-human IgG mouse antibody (Pharmingen, 08007E)was added to the plates and incubated. After O-phenylenediamine (OPD,Sumitomo Bakelite, ML-11300) was added as a peroxidase substrate, enzymeactivity was determined by absorbance measurement at 490 nm. The titerof the anti-HSA human IgG in the sera of the chimeric mice immunizedwith HSA increased after the immunization. On the other hand, controlICR mouse gave a background level of the anti-BSA human IgG titer afterthe immunization with HSA. The results are shown in FIG. 17. In FIG. 17,the number of days after the first immunization of the chimeric micewith HSA is plotted on the horizontal axis and the absorbance at 490 nmis plotted on the vertical axis. These results show that the antibodytiter of the antigen specific human IgG was increased by stimulationwith the HSA antigen in the chimeric mice retaining human chromosome #14fragments.

EXAMPLE 22 Detection of Human Antibody λ Chain in a Serum of the HumanChromosome #22 Fragment Transferred Chimeric Mouse

The chimeric mouse K22-7 from Example 3 (9 weeks old; 10% chimerism) wasbled and human antibody λ chain in the serum was detected by ELISA (seeExample 14). Ninety six-well microtiter plates were coated withanti-human antibody λ chain goat antibody (VECTOR LABORATORIES INC.,AI-3070) diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6) andthen a serum sample was added. Subsequently, biotin-labeled anti-humanantibody λ chain goat antibody (VECTOR LABORATORIES INC., BA-3070) wasadded to the plates and incubated. A complex of biotinylated horseradishperoxidase and avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC Kit)was added and incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) wasadded as a peroxidase substrate, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG antibody having λchain (Sigma, I-4014) was used as standard. The standard was dilutedstepwise with mouse serum-supplemented PBS. As a result, human antibodyλ chain was detected in the chimeric mouse at a concentrationcorresponding to 180 ng/ml of human IgG. These results show that humanantibody λ chain gene can function in the chimeric mouse retaining ahuman chromosome #22 fragment.

EXAMPLE 23 Detection of Human Antibody κ Chain in Sera of the HumanChromosome #2 Fragment Transferred Chimeric Mice

The chimeric mouse K2-8 from Example 13 (5 weeks old; 70% chimerism) andthe chimeric mice K2-3, K2-4 and K2-12 from Example 13 (9 weeks old;chimerisms was 50%, 20% and 80%, respectively) were bled and humanantibody κ chain in the sera was detected by ELISA (see Example 14).Ninety six-well microtiter plates were coated with anti-human antibody κchain goat antibody (VECTOR LABORATORIES INC., AI-3060) diluted with 50mM carbonate-bicarbonate buffer (pH 9.6) and then a serum sample wasadded. Subsequently, biotin-labeled anti-human antibody κ chain goatantibody (VECTOR LABORATORIES INC., BA-3060) was added to the plates andincubated. A complex of biotinylated horseradish peroxidase and avidinDH (VECTOR LABORATORIES, INC., Vectastain ABC Kit) was added andincubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzymeactivity was determined by absorbance measurement at 450 nm. Purifiedhuman IgG antibody having κ chain (Sigma, I-3889) was used as standard.The standard was diluted stepwise with mouse serum-supplemented PBS. Theresults are shown in Table 9.

TABLE 9 Concentration of Human Antibody κ Chain in Chimeric Mouse(ELISA) Chimeric Mouse Igκ (mg/l) K2-3 124 K2-4 85 K2-8 25 K2-12 56

The chimeric mice K2-3 and K2-4 retaining human chromosome #2 fragmentsfrom Example 13 were immunized with HSA, 3 times at days 66, 80 and 102after birth by the same method as in Example 20. The chimeric mouseK2-12 was immunized with HSA, 4 times at days 63, 77, 91 and 116 afterbirth by the same method as in Example 20. Human antibody κ chainagainst HSA in the sera of the chimeric mice was detected by ELISA (seeExample 14). Ninety six-well microtiter plates were coated with HSA(Sigma, A 3782) diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6)and then a sample was added. Subsequently, biotin-labeled anti-humanantibody κ chain goat antibody (VECTOR LABORATORIES, INC., BA-3060) wasadded to the plates and incubated. A complex of biotinylated horseradishperoxidase and avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC Kit)was added and incubated. After OPD (Sumitomo Bakelite, ML-11300) wasadded as a peroxidase substrate, enzyme activity was determined byabsorbance measurement at 490 nm. The titer of the anti-HSA human κchain in the sera of the chimeric mice immunized with HSA increasedafter the immunization. On the other hand, control ICR mouse gave abackground level of the anti-HSA human κ chain titer after theimmunization. The results are shown in FIG. 18. In FIG. 18, the numberof days after the first immunization of the chimeric mice with HSA isplotted on the horizontal axis and the absorbance at 490 nm is plottedon the vertical axis. These results show that human antibody κ chaingene can function in the chimeric mice retaining human chromosome #2fragments and that the antibody titer of the antigen specific human Ig κwas increased by stimulation with the HSA antigen in the chimeric mice.

EXAMPLE 24 Preparation of Human Antibody Heavy Chain (Chain or γChain)-Producing Hybridomas from the Human Chromosome #14 TransferredChimeric Mouse

The spleen was removed from the HSA-immunized chimeric mouse K9 (seeExample 21) at day 136 after birth. A spleen cell was fused with amyeloma cell to produce a hybridoma. The hybridoma was produced using amyeloma cell Sp-2/0-Ag14 (Dainippon Pharmaceutical Co., Ltd., 05-554) bythe method described in Toyama and Ando, “Monoclonal Antibody ExperimentProcedure Manual”, published by Kodansha Scientific, 1991. The cellswere inoculated into a medium containing 10% ORIGEN Hybridoma CloningFactor (HCF, Bokusui Brown) in eight 96-well plates and G418 was addedafter 3 days at a concentration of 1 mg/ml, followed by cultivation for1-3 weeks. The culture supernatant was analyzed by ELISA. Ninetysix-well microtiter plates were coated with anti-human μ chain mousemonoclonal antibody (Sigma, I-6385) diluted with 50 mMcarbonate-bicarbonate buffer (pH 9.6) and a sample diluted with PBS wasadded. Subsequently, peroxidase-labeled anti-human A chain mouseantibody (The Binding Site LIMITED, MP008) was added to the plates andincubated. 2,2′-Azino-di-(3-ethyl-benzothiazoline-6-sulfonate)diammonium salt (ABTS, Kirkegaard & Perry Laboratories Inc., 04-10-17)was used as a substrate to detect seven positive clones. In thedetection of Y chain-producing clones, 96-well microtiter plates werecoated with anti-human γ chain mouse monoclonal antibody (Sigma, I-6260)and a sample diluted with PBS was added. Subsequently,peroxidase-labeled anti-human γ chain mouse antibody (Pharmingen,08007E) was added to the plates and incubated. ABTS (Kirkegaard & PerryLaboratories Inc., 04-10-17) was used as a substrate and two humanantibody γ chain-positive clones were obtained.

EXAMPLE 25 Preparation of Human Antibody Light Chain-ProducingHybridomas from the Human Chromosome #2 Transferred Chimeric Mouse

The spleen was removed from the HSA-immunized chimeric mouse K2-3 (seeExample 23) at day 105 after birth. A spleen cell was fused with amyeloma cell to produce a hybridoma. The hybridoma was produced using amyeloma cell P3X63Ag8.653 (Dainippon Pharmaceutical Co., Ltd., 05-565)by the method described in Toyama and Ando, “Monoclonal AntibodyExperiment Procedure Manual”, published by Kodansha Scientific, 1991.The cells were inoculated into a medium containing 10% HCF (BokusuiBrown) in ten 96-well plates and G418 was added after 3 days at aconcentration of 1 mg/ml, followed by cultivation for 1-3 weeks. Theculture supernatant was assayed by ELISA. The ELISA analysis wasconducted by the same method as in Example 23 and two human antibody κchain-positive clones were obtained.

EXAMPLE 26 Re-Marking of the G418 Resistance-Marked Human Chromosome #22with Puromycin Resistance

The A9 cells retaining the G418 resistance-marked human chromosome #22(A9/#22 γ2) from Example 1 were re-marked with puromycin resistance bythe same method as in Example 16. About 200 colonies of double-drugresistant clones obtained by electroporation of the 72 cells withpPGKPuro were collected as one group and three such groups (P1, P2 andP3) were used as donor cells to perform microcell transfer intowild-type mouse A9 cells. As a result, 6, 1 and 3 of double-drugresistant clones were obtained from the groups P1, P2 and P3,respectively. The clone 6-1 from group P3 was used as a microcell donorcell and a wild-type A9 cell as a recipient cell to perform a microcelltransfer experiment (see Example 18). As a result, the human chromosome#22 was confirmed to have been further marked with a puromycinresistance gene. The preparation of microcells and the fusion with A9cells were conducted by the same methods as in Example 1. As a result,twenty eight G418 resistant colonies appeared 11 days after themicrocell transfer. After the medium for these colonies was changed toone containing 8 μg/ml puromycin, these colonies were cultured for 3days to give 21 (75%) viable colonies. In many cases of microcellfusion, only one or few chromosomes are transferred into a recipientcell. Hence, cotransfer of both the resistance genes at a high frequencyshows that the G418 resistance-labeled chromosome #22 retained in the6-1 clone was marked with the puromycin resistance gene.

EXAMPLE 27 Preparation and Sequencing of cDNA of a Human Antibody HeavyChain Variable Region from the Human Antibody Heavy Chain-ProducingHybridoma

Among the human antibody heavy chain (IgM)-producing hybridomas obtainedin Example 15, H4B7 (HSA-specific) and H8F9 (non-specific) hybridomaswere selected. Total RNAs were obtained from these hybridomas usingISOGEN (Nippon Gene). The synthesis of cDNA from 5 μg each of the totalRNAs was conducted with a Ready-To-Go T-primed 1st strand Kit (PharmaciaCo.). Using the resulting cDNA and the following primers prepared withreference to Larrick et al., BIO/TECHNOLOGY, 7, 934-, 1989; Word et al.,Int. Immunol., 1, 296-, 1989, PCR was performed to amplify a humanantibody heavy chain variable region.

CM1 (human IgM constant region): (SEQ ID NO:45) 5′-TTGTATTTCCAGGAGAAAGTGCM2 (ditto): (SEQ ID NO:46) 5′-GGAGACGAGGGGGAAAAGGG HS1 (human heavychain variable region): (SEQ ID NO:47) 5′-ATGGACTGGACCTGGAGG (AG) TC(CT) TCT (GT) C (a mixture of 8 sequences) HS2 (ditto): (SEQ ID NO:48)5′-ATGGAG (CT) TTGGGCTGA (GC) CTGG (GC) TTT (CT) T (a mixture of 16sequences) HS3 (ditto): (SEQ ID NO:49) 5′-ATG (AG) A (AC) (AC) (AT) ACT(GT) TG (GT) (AT) (GCT) C (AT) (CT) (GC) CT (CT) CTG (a mixture of 6144sequences)* ( ) means that any one of the bases therein should be selected.

In both cases of the H4B7 and H8F9 hybridomas, the first run of PCR wasperformed by using three kinds of primer combinations of HS1×CM1,HS2×CM1 and HS3×CM1 in cycles at 94° C. for 1 minute, 50° C. for 2minutes and 72° C. for 3 minutes with a Thermal Cycler 140 (Perkin-ElmerCorp.). The PCR products were amplified again under the same temperatureconditions in 30 cycles using HS1×CM2, HS2×CM2 and HS3×CM2 primers,respectively. The amplification products were electrophoresed on a 1.5%agarose gel and detected by staining with ethidium bromide. As a result,an amplification product of about 490 bp was detected with the HS3×CM2primer in the case of the H4B7 hybridoma. In the case of the H8F9hybridoma, a slight band was detected at the same site with the HS3CM2primer. The band in the case of H8F9 was amplified again with theHS3×CM2 primer in 30 cycles under the same temperature conditions asabove. As a result, the amplification product was detected as a veryintensive signal. These PCR products were cloned into a pBlueScriptIISK+ (Stratagene Ltd.) at a SmaI site in accordance with the methoddescribed in Ishida et al., “Gene Expression Experiment Manual”,published by Kodansha Scientific, 1995. Among the amplificationproduct-inserted plasmids, plasmids #2, #3, #4 (H4B7), #11, #13 and #14(H8F9) were selected and the nucleotide sequences of the amplificationproducts were determined with a Fluorescence Autosequencer (AppliedBiosystems Inc.). As a result of the comparison of the obtainednucleotide sequences or deduced amino acid sequences with those of knownhuman antibody VH region (Marks et al., Eur. J. Immunol. 21, 985-, 1991)and JH region (Ravetch et al., Cell, 27, 583-, 1981), it was revealedthat both the H4B7 and H8F9 hybridomas contained a combination of genesfor VH4 family and JH2. These results show that the chimeric mouseretaining human chromosome #14 partial fragment produced a completefunctional human antibody heavy chain protein.

EXAMPLE 28 Preparation and Sequencing of cDNA of Human Antibody κ Chainfrom the Spleen of the Human Antibody κ Chain-Expressing Chimeric Mouse

In the same manner as in Example 5, cDNA was prepared from the spleen ofthe chimeric mouse K2-8 from Example 13 which was confirmed to expresshuman antibody κ chain in Example 23. Using the resulting cDNA and thefollowing primers prepared with reference to Larrick et al.,BIO/TECHNOLOGY, 7, 934-, 1989; Whitehurst et al., Nucleic Acids Res.,20, 4929-, 1992, PCR was performed to amplify human antibody κ chainvariable region. cDNA from the liver of the chimeric mouse K2-8 and cDNAfrom the spleen of the chimeric mouse K3-2-2 derived from the TT2/#143-2 clone (see Example 10) were used as negative controls.

(SEQ ID NO: 50) KC2 (human Ig κ chain constant region):5′-CAGAGGCAGTTCCAGATTTC (SEQ ID NO: 51) KC3 (ditto):5′-TGGGATAGAAGTTATTCAGC (SEQ ID NO: 52) KVMIX (human Ig κ chain variableregion): 5′-ATGGACATG(AG)(AG)(AG)(AGT)(CT)CC(ACT)(ACG)G(CT)(GT)CA(CG)CTT (a mixture of 3456 sequences)* ( ) means that any one of the bases therein should be selected.

PCR was performed by using primer combinations of KVMIX×KC2 andKVMIX×KC3 in 40 cycles at 94° C. for 15 seconds, 55° C. for 15 secondsand 72° C. for 20 seconds with a Thermal Cycler 9600 (Perkin-ElmerCorp.). The amplification products were electrophoresed on a 1.5%agarose gel and detected by staining with ethidium bromide. As a result,expected amplification products of about 420 bp (KC2) and about 450 bp(KC3) were detected. In the case of the two negative controls, nospecific amplification product was detected. These amplificationproducts were cloned into a pBlueScriptII SK+ (Stratagene Ltd.) at aSmaI or EcoRI site in accordance with the method described in Ishida etal., “Gene Expression Experiment Manual”, published by KodanshaScientific, 1995. Among the amplification product-inserted plasmids,VK-#1 clone derived from the KVMIX×KC2 primers was selected and thenucleotide sequence of the amplification product was determined with aFluorescence Autosequencer (Applied Biosystems Inc.). Since the obtainednucleotide sequence did not contain a termination codon at any sitebetween an initiation codon and a constant region of human Igκ chain,the cloned amplification products are believed to encode a variableregion of functional human Igκ chain. As a result of the comparison ofthe obtained nucleotide sequences with those of known human antibody Vκregion (Klein et al., Eur. J. Immunol. 23, 3248-, 1993) and JK region(Whitehurst et al., supra), it was revealed that the VK-#1 clonecontained a combination of genes for Vκ3 family and Jκ4. These resultsshow that the chimeric mouse retaining human chromosome #2 partialfragment produced a complete functional human antibody κ chain protein.

EXAMPLE 29 Detection and Quantitation of Human Antibody γ ChainSubclasses and μ Chain in Sera of the Chimeric Mice Retaining HumanChromosome #14 Fragment

The chimeric mice K15A and K16A from Example 10 (derived from the 1-4clone, with chimerism of 70% and 50%, respectively) of 11 weeks afterbirth were bled and human antibody γ chain subclasses and μ chain in thesera were detected by the same ELISA method as in Example 14.

Quantative Determination of Human IgG1

Ninety six-well microtiter plates were coated with anti-human IgGantibody (Sigma, I-6260) diluted with PBS. A serum sample was added.Subsequently, peroxidase-labeled anti-human IgG1 antibody (Pharmingen,08027E) was added to the plates and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG1 antibody (Sigma,I-3889) was used as standard. The standard was diluted stepwise withmouse serum-supplemented PBS.

Quantative Determination of Human IgG2

Ninety six-well microtiter plates were coated with anti-human IgG2antibody (Sigma, I-9513) diluted with PBS. A serum sample was added.Subsequently, peroxidase-labeled anti-human IgG antibody (Sigma, A-0170)was added to the plates and incubated. After TMBZ (Sumitomo Bakelite,ML-1120T) was added, enzyme activity was determined by absorbancemeasurement at 450 nm. Purified human IgG2 antibody (Sigma, I-4139) wasused as standard. The standard was diluted stepwise with mouseserum-supplemented PBS.

Quantative Determination of human IgG3

Anti-human IgG3 antibody (Sigma, I-7260) was diluted with 100 mMglycine-HCl buffer (pH 2.5) and incubated for 5 minutes at roomtemperature, followed by 10-fold dilution with 100 mM phosphate buffer(pH 7.0).

Ninety six-well microtiter plates were coated with the anti-human IgG3antibody solution. A serum sample was added. Subsequently,peroxidase-labeled anti-human IgG antibody (Pharmingen, 08007E) wasadded to the plates and incubated. After TMBZ (Sumitomo Bakelite,ML-1120T) was added, enzyme activity was determined by absorbancemeasurement at 450 nm. Purified human IgG3 antibody (Sigma, I-4389) wasused as standard. The standard was diluted stepwise with mouseserum-supplemented PBS.

Quantative Determination of Human IgG4

Anti-human IgG4 antibody (Sigma, I-7635) was diluted with 100 mMglycine-HCl buffer (pH 2.5) and incubated for 5 minutes at roomtemperature, followed by 10-fold dilution with 100 mM phosphate buffer(pH 7.0).

Ninety six-well microtiter plates were coated with the anti-human IgG3antibody solution. A serum sample was added. Subsequently,peroxidase-labeled anti-human IgG antibody (Pharmingen, 08007E) wasadded to the plates and incubated. After TMBZ (Sumitomo Bakelite,ML-1120T) was added, enzyme activity was determined by absorbancemeasurement at 450 nm. Purified human IgG4 antibody (Sigma, I-4639) wasused as standard. The standard was diluted stepwise with mouseserum-supplemented PBS.

Quantative Determination of Human IgM

Ninety six-well microtiter plates were coated with anti-human μ chainmouse monoclonal antibody (Sigma, I-6385) diluted with PBS. A serumsample was added. Subsequently, peroxidase-labeled anti-human μ chainmouse antibody (The Binding Site Limited, MP008) diluted with mouseserum (Sigma, M5905)-supplemented PBS was added to the plates andincubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added as aperoxidase substrate, enzyme activity was determined by absorbancemeasurement at 450 nm. Purified human IgM having μ chain (CAPPEL,6001-1590) was used as standard. The standard was diluted stepwise withmouse serum (Sigma, M5905)-supplemented PBS.

The results are shown in Table 10. All the subclasses IgG1, IgG2, IgG3and IgG4, and IgM were detected in the two chimeric mice K15A and K16A.

TABLE 10 Concentrations of Human antibody IgG Subclasses and IgM in theChimeric Mice (ELISA) Chimeric mouse IgG1 IgG2 IgG3 IgG4 IgM (mg/l) K15A2.25 1.96 0.17 0.43 7.09 K16A 0.30 0.69 0.10 0.07 0.87

EXAMPLE 30 Preparation of Mouse ES Cell Clones (TT2) Retaining HumanChromosome #22

The cell clone 6-1 (A9/#22, G418 and puromycin resistant) from Example26 was used as a chromosome donor cell for the preparation of mouse EScell (TT2) retaining human chromosome #22. A wild-type TT2 cell line(see Example 9) was used as a chromosome recipient cell. The microcellfusion and the selection of puromycin resistant clones were conducted bythe same procedures as in the selection of G418 resistant clones inExample 9 except that the concentration of puromycin was 0.75 μg/ml. Thefrequency of the appearance of the puromycin resistant clones was 1-2per 10⁷ of TT2 cells. The puromycin resistant clones were stored frozenand genomic DNA was prepared by the same methods as in Example 2. Theretention of human chromosome #22 in the puromycin resistant clonePG22-1 was confirmed by the methods described in (1) and (2) below.

(1) PCR Analysis

Genomic DNA of the puromycin resistant clone was used as a template inPCR amplification. Among the genes on human chromosome #22 (GeneticMaps, supra), ten primers whose presence in the A9/#22 clone wasconfirmed in Example 2 were used in the PCR amplification. All themarkers which existed in the A9/#22 clone (see Example 2) were detected.

(2) Southern Blot Analysis

In accordance with the same method as described in Example 2 using humanL1 sequence as a probe, Southern blot analysis was conducted withgenomic DNAs obtained from wild-type TT2 (negative control), thechromosome donor cell 6-1 and the puromycin resistant TT2 cell clonePG22-1. The results are shown in FIG. 19. In FIG. 19, the molecularweights of DNAs are shown at the left side. The band pattern of thePG22-1 clone was equivalent to that of the 6-1 cell and the signalintensities were the same. Hence, it was confirmed that chromosome #22in the 6-1 cell had been transferred certainly into the PG22-1 clone.

These experiments demonstrate that the puromycin resistant TT2 cellclone PG22-1 retained the whole or the most part of human chromosome#22.

EXAMPLE 31 Production of Chimeric Mice from the Mouse ES Cells (TT2)retaining Human Chromosome #22

The cells in a frozen stock of the puromycin resistant TT2 cell clonePG22-1 from Example 30 which was confirmed to retain human chromosome#22 were thawed, started to culture and injected into 8-cell stageembryos obtained by mating ICR or MCH(ICR) male and female mice (CREAJAPAN, INC.); the injection rate was 10-12 cells per embryo. The embryoswere cultured in a medium for ES cells (see Example 9) overnight todevelop to blastocysts. Two and a half day after a foster mother ICRmouse was subjected to a pseudopregnant treatment, about ten of theinjected embryos were transplanted to each side of the uterus of thefoster mother.

The results are shown in Table 11.

TABLE 11 Production of chimeric mice from the TT2 cell clone retaininghuman chromosome #22 Number of ES ES cell Puromycin cell-injected Numberof Number Contribution clone/human resistant 8-cell stage offspringchimeric to coat color chromosome clone No. embryos mice mice <20%20-50% 50-80% TT2/#22 PG22-1 266 36 8 4 1 3

As a result of the transplantation of a total of 266 injected embryos,36 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 36produced offsprings, 8 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cells.

These results show that the ES cell clone (derived from TT2, PG22-1)retaining human chromosome #22 maintain the ability to produce chimera,that is, the ability to differentiate into normal tissues of mouse.

EXAMPLE 32 Detection and Quantitation of Human Antibody λ Chain In Seraof the Chimeric Mice Retaining Human Chromosome #22

The concentration of human antibody λ in the sera of the chimeric miceKPG22-1, 2 and 3 from Example 31 was determined by ELISA in accordancewith the same procedure as in Example 14. The chimeric mice of 2 monthsafter birth were bled and human antibody λ chain in the sera wasdetected by ELISA. Ninety six-well microtiter plates were coated withanti-human immunoglobulin λ chain antibody (VECTOR LABORATORIES INC.,IA-3070) diluted with PBS and then a serum sample was added.Subsequently, biotin-labeled anti-human immunoglobulin λ chain antibody(VECTOR LABORATORIES INC., BA-3070) was added to the plates andincubated. A complex of biotinylated horseradish peroxidase and avidinDH (VECTOR LABORATORIES, INC., Vectastain ABC Kit) was added andincubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzymeactivity was determined by absorbance measurement at 450 nm. Purifiedhuman IgM antibody having λ chain (Dainippon Pharmaceutical Co., Ltd.,U13200) was used as standard. The standard was diluted stepwise withmouse serum-supplemented PBS. The results are shown in Table 12. Theseresults show that human antibody λ chain gene can function in thechimeric mice retaining human chromosome #22.

TABLE 12 Concentration of Human Antibody λ Chain in Chimeric Mice(ELISA) Chimeric Mouse % Chimerism Igλ (mg/l) KPG22-1 50 12 KPG22-2 5018 KPG22-3 20 24

EXAMPLE 33 Detection of Anti-human HSA Human Antibody λ Chain in a Serumof the Human Chromosome #22 Transferred Chimeric Mouse

The chimeric mouse KPG22-3 from Example 31 was immunized with HSA, 3times at days 79, 94 and 110 after birth by the same method as inExample 20. Human antibody λ chain in the serum of the chimeric mousewas detected by ELISA in accordance with the same procedure as inExample 14. Ninety six-well microtiter plates were coated with HSA(Sigma, A 3782) diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6)to a concentration of 5 μg/ml and a serum sample was added. Biotinylatedanti-human Igλ antibody (VECTOR LABORATORIES INC., BA-3070) was added.Subsequently, a complex of biotinylated-horseradish peroxidase andavidin DH (VECTOR LABORATORIES, INC., Vectastain ABC Kit) was added tothe plates and incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) wasadded, enzyme activity was determined by absorbance measurement at 450nm. The titer of the anti-HSA human λ chain in the serum of the chimericmouse increased after the immunization. On the other hand, control ICRmouse gave a background level of the anti-HSA human λ chain titer afterthe immunization with HSA. The results are shown in FIG. 20. In FIG. 20,the number of days after the first immunization of the chimeric mousewith HSA is plotted on the horizontal axis and the absorbance at 450 nmis plotted on the vertical axis. These results show that human antibodyλ chain gene can function in the chimeric mouse retaining humanchromosome #22 and that the antibody titer of the antigen specific humanIgλ was increased by stimulation with the HSA antigen.

EXAMPLE 34 Preparation of Human Antibody Light Chain-ProducingHybridomas from the Human Chromosome #22 Transferred Chimeric Mouse

The spleen was removed from the mouse KPG22-3 (see Example 33) at day113 after birth by the same method as in Example 25. A spleen cell wasfused with a myeloma cell to produce a hybridoma. The hybridoma wasproduced using a myeloma cell SP-2/0-Ag14 (Dainippon Pharmaceutical Co.,Ltd., 05-554) by the method described in Toyama and Ando, “MonoclonalAntibody Experiment Manual”, published by Kodansha Scientific, 1991. Thecells were inoculated into a medium containing 10% HCF (Air Brown) infive 96-well plates and cultured for 1-3 weeks. The supernatant of theculture solution in colony-positive wells was analyzed by ELISA. TheELISA analysis was conducted by the same method as in Example 33 andfour human antibody λ chain-positive clones were obtained.

EXAMPLE 35 Preparation of Mouse ES Cell Clones Retaining Both a HumanChromosome #22 Partial Fragment and a Human Chromosome #14 PartialFragment

The 6-1 cell clone from Example 26 (A9/#22, G418 and puromycinresistant) was used as a chromosome donor cell for the preparation ofmouse ES cells retaining both a human chromosome #22 partial fragmentand a human chromosome #14 partial fragment. The G418 resistant TT2 cellclone 1-4 retaining a human chromosome #14 partial fragment from Example9 was used as a chromosome recipient cell. The experiment of microcellfusion and the selection of puromycin resistant cells were carried outby the same methods as in the selection of the G418 resistant clones inExample 9 except that the concentration of puromycin was 0.75 μg/ml. Asa result, the frequency of the appearance of the puromycin resistantclones was 1-2 per 10⁷ of 1-4 cells. The retention of G418 resistance inthe puromycin resistant clones was confirmed from the fact that theseclones were grown in the presence of 300 μg/ml G418. The double-drugresistant clones were stored frozen and genomic DNAs were prepared bythe same methods as in Example 2. The retention of human chromosome #22and a human chromosome #14 partial fragment in the double-drug resistantclone PG22-5 was confirmed by PCR analysis. With genomic DNA of thedouble-drug resistant clone used as a template, PCR amplification wasconducted using primers whose presence on chromosome #22 was confirmedin Example 2 (A9/#22) and primers whose presence on chromosome #14 wasconfirmed in Example 9 (TT2/#14 1-4); as a result, three markers(D22S275, D22S315 and Igλ) of the ten markers on chromosome #22 and allof the markers on chromosome #14 in the TT2/#14 1-4 clone were detected.

These experiments demonstrate that the obtained double-drug resistantTT2 cell clone retained both a human chromosome #22 partial fragment anda human chromosome #14 partial fragment.

EXAMPLE 36 Production of the Chimeric Mouse from the Mouse ES Cell CloneRetaining Both a Human Chromosome #22 Partial Fragment and a HumanChromosome #14 Partial Fragment

The cells in a frozen stock of the G418 and puromycin double-resistantTT2 cell clone PG22-5 from Example 35 which was confirmed to retain ahuman chromosome #22 partial fragment and a human chromosome #14 partialfragment were thawed, started to culture and injected into 8-cell stageembryos obtained by mating ICR or MCH(ICR) male and female mice (CREAJAPAN, INC.); the injection rate was 10-12 cells per embryo. The embryoswere cultured in a medium for ES cells (see Example 9) overnight todevelop to blastocysts. Two and a half day after a foster mother ICRmouse was subjected to a pseudopregnant treatment, about ten of theinjected embryos were transplanted to each side of the uterus of thefoster mother. The results are shown in Table 13.

TABLE 13 Production of the chimeric mouse from the mouse ES cell cloneretaining both a human chromosome #22 partial fragment and a humanchromosome #14 partial fragment Number of ES cell- ES cell Double-druginjected Number of Number of Contribution clone/human resistant 8-cellstage offspring chimeric to coat color chromosome clone No. embryos micemice <20% 20-50% 50-80% TT2/#22 + #14 PG22-5 302 16 5 3 2 0

As a result of the transplantation of a total of 302 injected embryos,16 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 16produced offsprings, 5 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cell.

These results show that the ES cell clone PG22-5 retaining a humanchromosome #22 partial fragment and a human chromosome #14 partialfragment maintains the ability to produce chimera, that is, the abilityto differentiate into normal tissues of mouse.

EXAMPLE 37 Detection of Human Antibody λ Chain and μ Chain In Sera ofthe Chimeric Mice Derived from the ES Cells Retaining Both a HumanChromosome #22 Partial Fragment and a Human Chromosome #14 PartialFragment

The chimeric mice KPG22-9, 10 and 12 from Example 36 were immunized withHSA. The chimeric mice KPG22-9 and 10 were immunized 11 weeks afterbirth and bled 2 weeks after the immunization. The chimeric mouseKPG22-12 was immunized twice at 7 and 11 weeks after birth and bled 2weeks after the second immunization.

A serum human antibody μ chain, a serum human antibody λ chain, and aserum antibody having both human antibody λ and A chains were detectedby ELISA in accordance with Example 14.

For the detection of complete human antibody molecules, 96-wellmicrotiter plates were coated with anti-human immunoglobulin λ chainantibody (Kirkegaard & Perry Laboratories Inc., 01-10-11) diluted withPBS and a serum sample was added. Subsequently, peroxidase-labeledanti-human immunoglobulin μ chain antibody (The Binding Site Limited,MP008) was added to the plates and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added as a peroxidase substrate, enzyme activitywas determined by absorbance measurement at 450 nm. Purified human IgMantibody having λ chain (Dainippon Pharmaceutical Co., Ltd., U13200) wasused as standard. The standard was diluted stepwise with mouseserum-supplemented PBS. Human antibody μ and λ chains were detected anddetermined quantitatively by ELISA in the same manner as in Examples 29and 32. The results are shown in Table 14.

TABLE 14 Concentrations of Human Antibodies in Chimeric Mice (ELISA)Chimeric Chimerism Igλ IgM, λ ES clone mouse (%) IgM (mg/l) (mg/l)(mg/l) PG22-5 KPG22-9 30 2.54 9.9 0.043 PG22-5 KPG22-10 5 4.96 21.50.333 PG22-5 KPG22-12 40 3.71 7.0 0.048 3-2 K9 50 6.66 — <0.003 PG22-1KPG22-2 50 — 17.6 <0.003

Both λ and μ chains were detected in the chimeric mice. An antibodymolecule having both human antibody μ and λ chains was detected. Theseresults show: the human antibody λ chain gene and human antibody μ chaingene can function at the same time in the chimeric mice derived from theES cells retaining human chromosome #22 partial fragments and humanchromosome #14 partial fragments; and a complete antibody containingboth human heavy and light chains was produced in part of the B cells.

The control mice, that is, the chimeric mouse K9 retaining only humanchromosome #14 from Example 10 and the chimeric mouse KG22-2 retainingonly human chromosome #22 from Example 31, gave background levels of anantibody having both human antibody λ and μ chains in the sera. It wasconfirmed that in these detection systems, only a complete antibodymolecule having human λ and μ chains was detected.

EXAMPLE 38 Detection of Human Antibody Having Human κ and μ Chains inSera of the Chimeric Mice Derived from the ES Cells Retaining Both HumanChromosome #2 Partial Fragments and Human Chromosome #14 PartialFragments

The chimeric mouse KPG-15 (derived from the TT2ES clone PG5, 10%chimerism) was immunized during 2-3 months after birth 3 times with 0.2ml of a solution of human serum albumin (HSA, Sigma, A3782) and adjuvant(MPL+TDM Emulsion, RIBI Immunochem Research Inc.) in PBS at a HSAconcentration of 0.25 mg/ml and bled (see Example 15). The chimericmouse KPG-26 (derived from the TT2ES clone PG6, 40% chimerism) of 6weeks after birth was bled. The concentration of a complete humanantibody molecule in the sera was determined by ELISA in accordance withExample 14. Ninety six-well microtiter plates were coated withanti-human immunoglobulin κ chain antibody (Kirkegaard & PerryLaboratories Inc., 01-10-10) diluted with PBS, and a serum sample wasadded. Subsequently, peroxidase-labeled anti-human immunoglobulin μchain antibody (The Binding Site Limited, MP008) was added to the platesand incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added as aperoxidase substrate, enzyme activity was determined by absorbancemeasurement at 450 nm. Purified human IgM antibody having κ chain(CAPPEL, 6001-1590) was used as standard. The standard was dilutedstepwise with mouse serum-supplemented PBS. The concentrations of κchain and μ chain were determined by the same method as in Example 20.The results are shown in Table 15.

TABLE 15 Concentrations of Human Antibodies in Chimeric Mice (ELISA)Chimeric Chimerism Igκ IgM, κ ES clone mouse (%) IgM (mg/l) (mg/l)(mg/l) PG-5 KPG15 10 0.18 1.01 0.075 PG-6 KPG26 40 1.52 1.26 0.018 3-2K9 50 6.66 — <0.002 5-1 K2-9 40 — 135 <0.002

A antibody molecule having both human antibody μ and κ chains wasdetected. The control mice, that is, the chimeric mouse K9 retainingonly human chromosome #14 from Example 10 and the chimeric mouse K2-9retaining only human chromosome #2 from Example 13, gave backgroundlevels (<0.002 mg/ml) of an antibody having human antibody κ and μchains in the sera. These results show: the human antibody κ chain geneand human antibody μ chain gene can function at the same time in thechimeric mice derived from the ES cells retaining both human chromosome#2 partial fragments and human chromosome #14 partial fragments; and acomplete antibody molecule containing both human heavy and light chainswas produced in part of the B cells.

EXAMPLE 39 Preparation of Mouse ES Cell Clone (TT2F, XO) Retaining aHuman Chromosome #2 Partial Fragment

The cell clone PG1 from Example 16 was used as a chromosome donor cellfor the preparation of a mouse ES cell (XO) retaining a human chromosome#2 partial fragment. A TT2F cell (purchased from Lifetec Oriental Co.)having a karyotype of (39, XO), which was reported to differentiateefficiently into an oocyte in chimeric mice (Shinichi Aizawa, “BiomanualSeries 8, Gene Targeting” published by Yodosha, 1995), was used as achromosome recipient cell. The experiment of microcell fusion and theselection of puromycin resistant cells were carried out by the samemethods as in the selection of the G418 resistant clones in Example 9except that the concentration of puromycin was 0.75 μg/ml. The frequencyof the appearance of the puromycin resistant clones was 5 per 1 of TT2Fcells. The puromycin resistant clones were stored frozen and genomicDNAs were prepared from the clones by the same methods as in Example 2.The retention of human chromosome #2 partial fragments in the drugresistant clones P-20 and P-21 was confirmed by PCR analysis. As aresult of PCR amplification using genomic DNAs of the drug resistantclones as templates and three kinds of primers Cκ, FABP1 and Vκ1-2 whosepresence in the A9/#2 W23 clone was confirmed in Example 1, all of thethree primers gave expected amplification products in both of the twoclones.

These experiments demonstrate that the obtained puromycin resistant EScell clone (TT2F, XO) retained a human chromosome #2 partial fragment.

EXAMPLE 40 Production of the Chimeric Mice from the Mouse ES Cell Clone(TT2F, XO) Retaining a Human Chromosome #2 Partial Fragment

The cells in a frozen stock of the puromycin resistant TT2F cell cloneP-21 from Example 39 which was confirmed to retain a human chromosome #2partial fragment were thawed, started to culture and injected into8-cell stage embryos obtained by mating ICR or MCH(ICR) male and femalemice (CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo.The embryos were cultured in a medium for ES cells (see Example 9)overnight to develop to blastocysts. Two and a half day after a fostermother ICR mouse was subjected to a pseudopregnant treatment, about tenof the injected embryos were transplanted to each side of the uterus ofthe foster mother. The results are shown in Table 16.

TABLE 16 Production of the chimeric mice from the TT2F cell cloneretaining a human chromosome #2 partial fragment Number of ES cell- EScell Puromycin injected Number of Number of Contribution clone/humanresistant 8-cell stage offspring chimeric to coat color chromosome cloneNo. embryos mice mice <20% 20-50% 50-90% 100% TT2F/#2fg. P-21 141 20 9 02 3 4

As a result of the transplantation of a total of 141 injected embryos,20 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2F cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 20produced offsprings, 9 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cell. Four of the 9mice were chimeric mice having a full agouti coat color from the EScells.

These results show that the ES cell clone P-21 retaining a humanchromosome #2 partial fragment maintains the ability to produce chimera,that is, the ability to differentiate into normal tissues of mouse.

EXAMPLE 41 Detection and Quantitative Determination of Human Antibody κChain In Sera of the Chimeric Mice Derived from the TT2F Clone Retaininga Human Chromosome #2 Partial Fragment

The chimeric mice K2-1F, 2F, 3F and 4F (derived from the P-21 clone,100% chimerism) from Example 40 of about 1 month after birth were bledand the concentration of human antibody κ chain in the sera wasdetermined quantitatively by ELISA in the same manner as in Example 20.

The results are shown in Table 17. It was confirmed that the humanantibody κ chain gene could function in the chimeric mice when the TT2Fwas used as an ES cell.

TABLE 17 Concentration of Human Antibody κ Chain in Chimeric Mice(ELISA) Chimeric mouse % Chimerism Igκ (mg/l) K2-1F 100 66 K2-2F 100 156K2-3F 100 99 K2-4F 100 20

EXAMPLE 42 Confirmation of the Retention of Human Chromosome inProgenies of the Chimeric Mice Derived from the Mouse ES Cell (TT2F, XO)Retaining a Human Chromosome #2 Partial Fragment

Examination was made as to whether ES cell-derived progenies would bereproduced by mating the female chimeric mice K2-1F and K2-4F (both wereof 100% chimerism in coat color) from Example 40 with ICR male mice. Insuch a mating, offspring mice of an agouti coat color should bereproduced from the oocytes derived from the TT2F cell (agouti coatcolor, dominant) in the chimeric mice and offspring mice of an albinocoat color should be reproduced from oocytes derived from ICR if theoocytes are fertilized with the sperms from ICR male mice (albino,recessive). All the viable offspring mice (K2-1F, 10 mice and K2-4F, 5mice) obtained by one mating of the respective combinations had anagouti coat color which derived from the ES cells. The retention ofhuman chromosome fragments in genomic DNAs prepared from the tails ofthe offspring mice was examined by a PCR method. As a result of the PCRamplification using three kinds of primers whose presence in the P-21clone (see Example 39) was confirmed, the presence of these threemarkers was confirmed in 4 out of the ten mice from K2-1F and in 2 outof the five mice from K2-4F. The results of the PCR of these 15offspring mice are shown in FIG. 21. In FIG. 21, markers (φX174/HaeIIIfragment, Nippongene) and the DNA molecular weights of main bands areshown at the right side and the lengths of expected products ofamplification with the respective primers are shown by arrows at theleft side. At the right side, the results with tail-derived DNA of themother chimeric mice K2-1F and K2-4F (positive controls) are shown.These results show that the TT2 cell clone P-21 differentiated intofunctional oocyte in the chimeric mice and that a human chromosome #2partial fragment was transmitted to offsprings through oocyte.

EXAMPLE 43 Confirmation of the Retention of Human Chromosome inProgenies of the Chimeric Mice Derived from the Mouse ES Cell (TT2, XY)Retaining a Human Chromosome #2 Partial Fragment

Examination was made as to whether ES cell-contributed offspring micewould be produced by mating K2-18 (70% chimeric male mouse from Example13) with K2-19 (60% chimeric female mouse of Example 13) or non-chimericfemale littermates. Since TT2 cell has the karyotype of (40, XY), it maydifferentiate into a functional sperm in the male chimeric mouse K2-18.If this is the case, offspring mice of an agouti coat color should bereproduced from ICR (albino, recessive)-derived oocytes fertilized withsperms from TT2 cell (agouti color, dominant) in the chimeric mice.While a total of 110 viable offspring mice were obtained by the mating,ten had an agouti coat color which derived from the ES cells. Theretention of human chromosome fragments in genomic DNAs prepared fromthe tails of 7 out of the ten offspring mice of an agouti coat color wasexamined by a PCR method. As a result of PCR amplification using twokinds of primers Cκ and FABP1 whose presence in the 5-1 clone (TT2/#2fg.Example 12) was confirmed and primer Vκ1-2 which was shown in Example 1,the presence of all of the three markers was confirmed in 2 out of theseven mice. These results show that the TT2 cell clone 5-1 retaining ahuman chromosome #2 partial fragment differentiated into functionalsperms in the chimeric mice and that the human chromosome #2 partialfragment was transmitted to offsprings through the sperms.

EXAMPLE 44 Detection and Quantitative Determination of Human Antibody κChain in Sera of Offspring Mice of the Chimeric Mice

The concentration of human antibody κ chain in the sera of the offspringmice K2-1F-1-10 and K2-4F-1˜5 from Example 42 was determinedquantitatively by ELISA. The mice of about 4-6 months after birth werebled and the concentration of human antibody κ chain in the sera wasdetermined by ELISA in the same manner as in Example 20.

The results are shown in Table 18 together with the data obtained inExample 42 on the retention of chromosome. It was confirmed that thehuman antibody K chain gene can function in the offspring micereproduced from the chimeric mice.

TABLE 18 Concentration of Human Antibody κ Chain in Offspring Mice(ELISA) Presence of Mother Number of human chromosome mouse mouse #2fragments Igκ (mg/l) K2-1F #1 − 0.58 K2-1F #2 + 84.1 K2-1F #3 + 12.8K2-1F #4 + 15.1 K2-1F #5 − 0.52 K2-1F #6 − 0.58 K2-1F #7 − 1.30 K2-1F #8− 0.90 K2-1F #9 − 0.56 K2-1F #10 + 28.8 K2-4F #1 − <0.04 K2-4F #2 + 23.3K2-4F #3 + 11.8 K2-4F #4 − 0.08 K2-4F #5 − 0.06

EXAMPLE 45 Analysis of Spleen Cells from the Human Chromosome #14Partial Fragment Transferred Chimeric Mice

Flow cytometry analysis was accordance with the method described in “NewBiochemical Experiment Lecture 12, molecular immunology I-ImmunocellsCytokines-”, edited by the Japanese Biochemical Society, 1989, publishedby Tokyo Kagaku Dojin; “Cell Technology Separated Volume 8, New CellTechnology Experiment Protocol”, edited by the University of Tokyo,Medical Science Institute, Anti-cancer Laboratory, 1991, published byShujunsha; and A. Doyle and J. B. Griffiths, “Cell & Tissue Culture:Laboratory Procedures”, published by John Wiley & Sons Ltd., 1996. Thespleen was removed from the chimeric mouse KPG06 (derived from the PG16clone, 30% chimerism) from Example 19 of six months after birth andtreated with an aqueous solution of ammonium chloride. The spleen cellswere stained with fluorescein isothiocyanate (FITC)-labeled anti-mouseCD45R (B220) antibody (Pharmingen, 01124A) in PBS containing 1% ratserum. After being washed, the cells were reacted with 0.1 μg ofbiotin-labeled anti-human IgM antibody (Pharmingen, 08072D) or a controlbiotin-labeled anti-human λ chain antibody (Pharmingen, 08152D) in PBScontaining 5% mouse serum and stained with 0.1 μg ofstreptoavidin-phycoerythrin (Pharmingen, 13025D), followed by analysiswith a flowcytometer (Becton Dickinson Immunocytometry Systems,FACSort). An ICR mouse retaining no human chromosome was used as acontrol for analysis by the same method. The results are shown in FIG.22. In FIG. 22, the human IgM is plotted on the horizontal axis and theCD45R (B220) is plotted on the vertical axis. A population of cellspositive to both B cell marker CD45R (FITC) and human IgM (PE) increasedby 4%, indicating that cells expressing human antibody μ chain on thecell surfaces were present in the chimeric mice.

EXAMPLE 46 Cloning and Sequencing of Variable Regions of Human AntibodyGenes from cDNA Derived from the Spleen of the Chimeric Mice ExpressingHuman Antibody Heavy Chain, κ and λ Chains, Respectively

In the same manner as in Example 5, cDNAs were synthesized from RNAsextracted from the spleens of the chimeric mice K15A (derived from the1-4 clone, prepared by the method described in Example 10), K2-8prepared in Example 13 and KPG22-2 prepared in Example 31, all of whichwere confirmed to express human antibody heavy chain, κ and λ chains inExamples 29, 23 and 32, respectively. PCR was performed using therespective cDNAs and the following primers to amplify the variableregions of respective human antibody. cDNA derived from the spleen of anon-chimeric mouse ICR was used as a negative control. Those primers setforth below without indication of reference literature were designed onthe basis of the nucleotide sequences obtained from data bases such asGenebank and the like.

K15A (heavy chain) For constant region: (SEQ ID NO:53) HIGMEX1-2:5′-CCAAGCTTCAGGAGAAAGTGATGGAGTC (SEQ ID NO:54) HIGMEX1-1:5′-CCAAGCTTAGGCAGCCAACGGCCACGCT (used in 2nd PCR of VH3BACK)For variable region:VH1/5BACK (59° C. 35 cycles, Marks et al., Eur. J. Immunol., 21, 985-,1991),VH4BACK (59° C., 35 cycles, Marks et al., supra), andVH3BACK (1st PCR: 59° C., 35 cycles; 2nd PCR: 59° C., 35 cycles, Markset al., supra)

K2-8 (light chain κ)

For constant region: KC2H 5′-CCAAGCTTCAGAGGCAGTTCCAGATTTC (SEQ ID NO:55)For variable region:Vk1/4BACK (55° C. 40 cycles, Marks et al., Eur. J. Immunol., 21, 985-,1991),Vk2BACK (55° C. 40 cycles, Marks et al., supra), andVk3BACK (55° C. 40 cycles, Marks et al., supra)

KPG22-2 (light chain λ)

For constant region:CλMIX (a mixture of the following three kinds of primers at an equalmolar ratio)

IGL1-CR: 5′-GGGAATTCGGGTAGAAGTCACTGATCAG (SEQ ID NO:56) IGL2-CR:5′-GGGAATTCGGGTAGAAGTCACTTATGAG (SEQ ID NO:57) IGL7-CR:5′-GGGAATTCGGGTAGAAGTCACTTACGAG (SEQ ID NO:58)For variable region:Vλ1LEA1 (55° C., 40 cycles, Williams et al. Eur. J. Immunol., 23, 1456-,1993),Vλ2MIX (55° C. 40 cycles, a mixture of Vλ2 LEA1 and Vλ2 JLEAD (Williamset al. (supra)) at an equal molar ratio)Vλ3MIX (55° C. 40 cycles, a mixture of Vλ3LEA1, Vλ 3JLEAD and Vλ3BACK4,which were reported in Williams et al. (supra) at an equal molar ratio.

The PCR was performed with combinations of the primers for constantregions with those for variable regions (3 primer pairs each for heavychain, κ and λ chains) at 94° C. for 15 seconds, at the annealingtemperatures shown with respect to the respective primers for variableregion for 15 seconds, at 72° C. for 20 seconds in the cycle numbersshown with respect to the respective primers for variable region using aThermal Cycler 9600 (Perkin-Elmer Corp.). In the second run of PCR usingVH3BACK, the amplification products of the first run of PCR wereamplified again with a combination of the two primers H1GMEX1-1 andVH3BACK. All of the amplification products were electrophoresed on a1.5% agarose gel and stained with ethiduim bromide for detection. As aresult, the amplification products having expected lengths (heavy chain,about 470 bp; light chain κ, about 400 bp; and light chain λ, about 510bp) were detected in all of the combinations. In the negative control,specific amplification product was not detected at the same position inany of the combinations. The obtained amplification products wereextracted from the agarose gel using prep.A.gene (Bio-Rad Laboratories,Inc.), treated with restriction enzymes (heavy chain, HindIII and PstI;light chain κ, HindIII and PvuII; and light chain λ, HindIII and EcoRI),and cloned into pUC119 (TAKARA SHUZO CO., LTD.) at the sites ofHindIII/PstI (heavy chain), HindIII/HincII (κ chain) and HindIII/EcoRI(λ chain). The nucleotide sequences of the products that were amplifiedwith the following primers and which were cloned into the plasmids weredetermined with a Fluorescence Autosequencer (Applied Biosystems Inc.).

HIGMEX1-2×VH1/5BACK: 10 clones

HIGMEX1-2×VH4BACK: 8 clones

HIGMEX1-2 (2nd PCR, HIGMEX1-1)×VH3BACK: 5 clones

KC2H×Vκ1/4BACK: 6 clones

KC2H×Vκ2BACK: 7 clones

KC2H×Vκ3BACK: 4 clones

CλMIX×Vλ1LEA1: 5 clones

CλMIX×Vλ2MIX: 6 clones

CλMIX×Vλ3MIX: 5 clones

The obtained nucleotide sequences were analyzed with DNASIS (HitachiSoftware Engineering Co., Ltd.). The results show that all of thesequences were derived from human and that they were functionalsequences which did not contain a termination codon at any site betweenan initiation codon and a constant region: this was true with all of theκ and λ chains and with 21 out of a total of 23 heavy chains. When thesame sequences were removed from the determined sequences, uniquevariable region sequences were identified as follows: 17 heavy chains,11κ chains, and 12λ chains.

EXAMPLE 47 Analysis of the Nucleotide Sequences of Variable Region ofHuman Antibody Genes from cDNA Derived from the Spleen of the ChimericMouse Expressing Human Antibody Heavy Chain, κ and λ Chains,Respectively

The nucleotide sequences determined in Example 46 (heavy chain, 17clones; κ chain, 11 clones; and λ chain, 12 clones) were analyzed in thefollowing points.

1. Identification of known germ line V gene segments used in therespective variable regions2. Identification of known germ line J gene segments used in therespective variable regions3. Identification of known germ line D gene segments used in the heavychain variable regions4. Identification of the addition of N region in the heavy chainvariable regions on the basis of the results of 1, 2 and 35. Determination of the amino acid sequences deduced from the nucleotidesequences of the respective variable regions

The results are shown in Table 19. For the identification in points of 1and 2, search for homology with germ line V and J segments registered inGenbank and the like was conducted with DNASIS. The VH segments, Vκsegments and Vλ segments are shown in Table 19 together with the familynames of the respective V fragments in accordance with the conventionsdescribed in Cook et al., Nature genetics, 7, 162-, 1994 (VH fragments),Klein et al., Eur. J. Immunol, 23, 3248-, 1993 (Vκ fragments) andWilliams et al. (supra) (Vλ fragments), respectively. For theidentification in point 3, search for homology with germ line Dfragments reported in Ichihara et al., The EMBO J., 7, 13, 4141-, 1988was conducted with DNASIS. Assignment was based on at least 8 bpidentity and the results are shown in Table 19. DN1* is believed to bethe new DN family segment reported in Green et al., Nature Genetics, 7,13-, 1994. For the identification in point 4, the nucleotide sequenceswhich did not appear in any germline sequences were determined to be Nregions on the basis of the results for 1(V), 2(J) and 3 (D). As aresult, N region was observed in 11 of the 13 sequences in which Dsegment was identified and its average length was 8.7 bp. For thedetermination in point 5, the respective sequences were converted byDNASIS to amino acid sequences which were expressed with one lettersymbols. In Table 19, only CDR3 region is shown. At the right side ofTable 19, the names of the primers used in cloning of the respectivevariable regions and the names of clones are shown.

TABLE 19 V family V segment CDR3 J (D) V primer Clone K15A VH1 VH1-8VRSSSWYEYYYYGMDV J6 (DN1) VH4BACK H4-10 VH1-18 GGTTMVRGLITTDWYFDL J12(DXP′1) VH1/5BACK H1-7 VH1-24 APYSGRFDY J4 (DK1) VH1/5BACK H1-6 VH1-16ERYYGSGSYQDYYYYYGMDV J6 (DXP′1) VH1/5BACK H1-2 VH1-16 GGYSGYEDYYYYGMDVJ6 (DK1) VH1/5BACK H1-10 VH2 VH2-5 SYFDWPDFDY J4 (DXP1) HV4BACK H4-14VH3 VH3-21 EGCSGGSCLPGYYYYGMDV J6 (DLR2) VH1/5BACK H1-4 VH2-23 AHGDPYFDYJ4 VH1/5BACK H1-3 VH3-23 DADAFDI J3 VH1/5BACK H1-8 VH3-23 SGWDY J4(DN1*) VH3BACK H3-3 VH3-23 TGFDL J2 VH4BACK H4-4 VH3-33EGGYGSVGDYYYYGMDV J6 (DXP′1) VH1/5BACK H1-9 VH3-33 GGYSYGYDYYYYGMDV J6(DXP′1) VH3BACK H3-5 VH3-33 GYSSGWYDY J4 (DN1*) VH4BACK H4-9 VH4 VH4-34RYSSGWYYFDY J4 (DN1*) VH4BACK H4-15 VR4-59 GRIAVASFDY J4 (DN1*) VH4BACKH4-2 VH4-59 GSGSYFHFDY J4 VH4BACK H4-6 K2-8 V κ 1 O18-8 QQHDNLPFT J3 Vκ 1BACK K1-1 O18-8 QQYDNLPIT J5 V κ 1BACK K1-3 O18-8 QAHDNLPFA J3 Vκ 2BACK K2-2 L1 QQYNSYPLT J4 V κ 2BACK K1-6 V κ 2 A17 MQGTHLLT J4 Vκ 2BACK K2-1 A17 MQGTHWIT J5 V κ 2BACK K2-5 V κ 3 A27 QQYGSSPTWT J1 Vκ 3BACK K3-1 A27 QQYGSSPFT J3 V κ 3BACK K3-4 A27 QQYGSSPLWT J1 V κ 3BACKK3-5 A27 QQYGSSPPWT 11 V κ 3BACK K3-6 V κ 6 A26-10 HQSSSLPQT J1 Vκ 2BACK K2-4 KPG22-2 V λ 1 DPL3 AAWDDSLDVV JC3 V λ 1LEA1 L1-3 DPL5GTWDSSLSAGV JC2 V λ 1LEA1 L1-4 DPL5 GTWDSSLSAGVV JC3 V λ 1LEA1 L1-6 DPLSGTWDSSLSAVV JC2 V λ 1LEA1 L1-9 DPLS QSYDSSLSGVV JC3 V λ 1LEA1 L1-8 V λ 2DPL10 CSYAGSSTLV JC2 V λ 2MIX L2-4 DPL11 SSYTSSSTVV JC2 V λ 2MIX L2-1DPL11 SSYTSSSTLV JC2 V λ 2MIX L2-3 DPL11 CSYTSSSTFV JC2 V λ 2MIX L2-7DPL12 SSYAGSNNLV JC3 V λ 2MIX L2-5 DPL12 SSYAGSNNFVV JC3 V λ 2MIX L2-6 Vλ 3 DPL16 NSRDSSGNLV JC2 V λ 3MIX L3-1

EXAMPLE 48 Preparation of a Targeting Vector for Knocking Out AntibodyGenes (Heavy-Chain and Light-Chain κ Genes) in TT2F ES Cells

It becomes possible to transfer a human chromosome #14 fragment markedwith a G418 resistance gene (Example 9) and human chromosome #2 (Example18) or #22 (Example 35) marked with a puromycin resistance gene into TT2(or TT2F) cells in which mouse antibody genes (heavy-chain, light-chainκ) are disrupted. Those chimeric mice which are produced from thesehuman chromosomes #14+#2 or #14+#22-transferred, mouse antibody genes(heavy-chain, light-chain κ)-disrupted TT2 (or TT2F) ES cells accordingto the method of Example 19 (heavy-chain+κ chain) or Example 36(heavy-chain+λ chain) are expected to produce antibodies both heavy- andlight-chains of which are mainly derived from humans. The abbreviationsof the restriction enzymes, etc. appearing in FIGS. 23-27 are asfollows.

Restriction enzymes: Kp: KpnI, B: BamHI, Bg2:BglII, RI: EcoRI, RV:EcoRV, N: Not, Sl: SalI, Sca: ScaI, Sfi: SfiI, Sm: SmaI, X: XhoI, (X):XhoI restriction site from λ vectordK: deletion of KpnI restriction site, dX: deletion of XhoI restrictionsite,(Sm/Sl): The SalI restriction site was changed to a blunt end andsubsequent ligation to a SmaI restriction site, (Sl/RV): The SalIrestriction site was changed to a blunt end and subsequent ligation toan EcoRV restriction site, Dotted portion: pBluescript SKII(+) or pUC18plasmid DNA

LoxP Sequence

1. Preparation of Plasmid pLoxP-STneo in which LoxP Sequence is Insertedat Both the Ends of a G418 Resistance Gene

For the deletion of a G418 resistance gene after knocking out anantibody gene of TT2F cells, it is necessary to insert LoxP sequence(Sauer et al., Proc. Natl. Acad. Sci. USA, 85, 5166-, 1988) which is therecognition sequence of Cre recombinase (Sauer et al., supra) at boththe ends of the G418 resistance gene (Example 1) in the same direction.Briefly, a G418 resistance cassette (STneo) was cut out from pSTneoBplasmid DNA (Example 1, Katoh et al., Cell Struct. Funct., 12:575, 1987;Japanese Collection of Research Biologicals (JCRB), Deposit Number:VE039) with restriction enzyme XhoI. The DNA fragment was purified byagarose gel electrophoresis and then blunted with T4-DNA polymerase(Blunting End Kit from Takara Shuzo). LoxP sequence-containing plasmidDNA pBS246 (Plasmid pBS246, loxP2 Cassette Vector, U.S. Pat. No.4,959,317) was purchased from GIBCO BRL. XhoI linker DNAs were insertedinto the EcoRI and SpeI restriction sites of this plasmid to change thesequences of these sites to a XhoI recognition sequence. The STneo DNAfragment described above was inserted into the EcoRV restriction site ofthe thus modified pBS246 to give plasmid pLoxP-STneo (FIG. 23).

2. Isolation of Genomic DNA Clones Containing C57BL/6-Derived AntibodyHeavy-Chain CA (IgM Constant Region) or Light-Chain Jκ-Cκ (Igκ JointRegion and Constant Region)

Since TT2 (or TT2F) cells were derived from F1 mice between C56BL/6 miceand CBA mice, the inventors have decided to prepare vectors for antibodygene knockout using genomic DNA clones derived from a C57BL/6 mouse. Asa genomic DNA library, an adult C57BL/6N male liver-derived λ DNAlibrary from Clontech was used. As a probe for screening, the followingsynthetic DNA sequences (60 mers) were used.

Heavy-chain C μ probe: (SEQ ID NO:59) 5′-ACC TTC ATC GTC CTC TTC CTC CTGAGC CTC TTC TAC AGC ACC ACC GTC ACC CTG TTC AAG-3′ Light-Chain κ probe:(SEQ ID NO:60 ) 5′-TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATC CAGTGA GCA GTT AAC ATC TGG AGG-3′

The λ clones were isolated and analyzed to subclone those DNA fragmentscontaining heavy-chain Cμ or light-chain Jκ-Cκ into plasmid pBluescriptSKII(+) (Stratagene) (heavy-chain Cμ: FIG. 24; light-chain Jκ-Cκ: FIG.25). These DNA fragments were used to prepare targeting vectors fordisrupting mouse antibody genes in TT2 (or TT2F) cells as describedbelow.

3. Preparation of a Vector Plasmid for Disrupting a Mouse AntibodyHeavy-Chain Gene

In the Cμ-encoding region in the genomic DNA fragment containing a mouseantibody heavy-chain constant region which was prepared in 2 above, aDNA fragment containing the 2nd to 4th exons (BamHI-XhoI) was replacedwith the LoxP-STneo gene prepared in 1 above (FIG. 26). The direction oftranscription of STneo was the same as that of the antibody heavy-chaingene. Further, a DT-A cassette A (Proc. Natl. Acad. Sci. USA, 87,9918-9922, 1990, made by Oriental Yeast Co.) (hereinafter referred to as“DT”) modified by changing the ApaI and SalI sites to NotI sites wasinserted into the NotI site of this plasmid. A plasmid DNA containing aDT gene having the same direction of transcription as that of theheavy-chain gene was selected. This plasmid DNA was amplified using E.coli DH5 and purified by cesium chloride equilibrium centrifugation(“Introduction to Cell Technology Experimental Operations”, published byKodansha, 1992). The purified plasmid DNA was cleaved at one site withrestriction enzyme SacII and used for transfection or TT2F ES cells. Asa probe for Southern blot analysis of transformant genomic DNA to detectfrom transformant TT2F ES cells those clones in which homologousrecombination has taken place in the antibody heavy-chain portion withthe targeting vector, a DNA fragment (about 500 bp) of the switch regionlocated upstream of Cμ-encoding region. This DNA fragment was obtainedby amplifying 129 mouse genomic DNAs by PCR under the followingconditions.

Sense primer: 5′-CTG GGG TGA GCC GGA TGT TTT G-3′ (SEQ ID NO:59)Antisense primer: 5′-CCA ACC CAG CTC AGC CCA GTT C-3′ (SEQ ID NO:62)Template DNA: 1 μg of EcoRI-digested 129 mouse genomic DNAsThe reaction buffer, deoxynucleotide mix and Taq DNA polymerase usedwere from Takara Shuzo.Reaction conditions: 94° C., 3 min, 1 cycle 94° C., 1 min; 55° C., 2min; 72° C., 2 min; 3 cycles 94° C., 45 sec; 55° C., 1 min; 72° C., 1min; 36 cycles

After it was confirmed that amplified DNA fragment can be cleaved at onesite with restriction enzyme HindIII as indicated in the Genbankdatabase, this DNA fragment was subcloned into the EcoRV restrictionsite of plasmid pBluescript. This plasmid DNA (S8) was cleaved withrestriction enzymes BamHI and XhoI. A PCR fragment (about 550 bp) waspurified by agarose gel electrophoresis to give a probe. Genomic DNAfrom those TT2F ES cells transformed with the targeting vector wasdigested with restriction enzymes EcoRI and XhoI, and separated byagarose gel electrophoresis. Then, Southern blotting was performed usingthe above probe.

4. Preparation of a Vector for Disrupting the Mouse Antibody Light-Chainκ Gene

The genomic DNA fragment prepared in 2 above contains the J region andconstant region of mouse antibody light-chain κ. A DNA fragment(SacII-BglII) containing the C region was replaced with the LoxP-STneogene having the disrupted KpnI site (FIG. 27). The LoxP-STneo gene wasprepared in 1 above. The direction of transcription of STneo wasopposite to that of the antibody gene. This plasmid DNA was constructedas follows:

the sequence of a multi-cloning site (EcoRI-HindIII) in plasmid pUC18was changed to the following sequences prepared by DNA chemicalsynthesis.

(SEQ ID NO:63) 5′-AATTCCCGCGGGTCGACGGATCCCTCGAGGGTACCA-3′ (SEQ ID NO:64)3′-GGGCGCCCAGCTGCCTAGGGAGCTCCCATGGTTCGA-5′ EcoRI SacII SalI BamHI XhoIKpnI HindIII

The EcoRI-SacII DNA fragment containing Jκ (FIG. 25) was inserted intothe EcoRI and SacII sites of this plasmid. Then, a 3′-endBglII-BglII-BglII-XhoI DNA fragment (FIG. 25) was inserted into theBamHI and XhoI sites of the resulting plasmid. A XhoI-SalI DNA fragmentcontaining DT and a XhoI DNA fragment containing LoxP-STneo having thedisrupted KpnI fragment were inserted sequentially into the XhoI andSalI sites of this plasmid. The direction of transcription of the DTgene is the same as that of the light-chain κ gene. This plasmid DNA wasamplified using E. coli DH5 and purified by cesium chloride equilibriumcentrifugation. The purified plasmid DNA was cleaved at one site withrestriction enzyme KpnI and used for transfection of TT2F ES cells. As aprobe for Southern blot analysis of transformant genomic DNA to detectfrom transformant TT2F ES cells those clones in which homologousrecombination has taken place in the antibody light-chain portion withthe targeting vector, a DNA fragment at the 3′ end of the light-chainJκ-Cκ genomic DNA fragment (see FIG. 25) (XhoI-EcoRI; about 1.4 kbp) wasused. Genomic DNA from those TT2F ES cells transformed with thetargeting vector was digested with restriction enzyme EcoRI, andseparated by agarose gel electrophoresis. Then, Southern blotting wasperformed using the above probe.

EXAMPLE 49 Production of a Mouse ES Cell Antibody Heavy-ChainGene-Disrupted Clone

In order to obtain a recombinant in which an antibody heavy-chain genehas been disrupted by homologous recombination (hereinafter, referred toas an “antibody heavy-chain homologous recombinant”), the antibodyheavy-chain targeting vector prepared in Section 3, Example 48 waslinearized with restriction enzyme SacII (Takara Shuzo), and transferredinto mouse TT2F ES cells according to the method described by ShinichiAizawa, “Biomanual Series 8, Gene Targeting”, published by Yodosha,1995. The TT2F cells were treated with trypsin and suspended in HBS at aconcentration of 2.5×10⁷ cells/ml. To the cell suspension, 5 μg of DNAwas added. Then, electroporation was performed with a gene pulser(Bio-Rad Laboratories, Inc.; resistor unit not connected) A voltage of250 V was applied at a capacitance of 960 μF using an electroporationcell of 4 mm in length at room temperature. The electroporated cellswere suspended in 20 ml of an ES medium and inoculated into two tissueculture plastic plates (Corning) of 100 mm into which feeder cells wereseeded preliminarily. Similarly, experiments using 10 and 15 μg of DNAwere also conducted. After one day, the medium was replaced with amedium containing 300 μg/ml of G418 (GENETICIN; Sigma). Seven to ninedays thereafter, a total of 176 colonies formed were picked up. Eachcolony was grown up to confluence in a 12-well plate, and then fourfifths of the culture was suspended in 0.2 ml of a preservation medium[ES medium+10% DMSO (Sigma)] and stored frozen at −80° C. The remainingone fifth was inoculated into a 12-well gelatin coated plate andcultured for 2 days. Then, genomic DNA was obtained from the cells(10⁶-10⁷ cells) with Puregene DNA Isolation Kit (Gentra System Co.).These genomic DNAs from G418 resistant TT2F cells were digested withrestriction enzymes EcoRI and XhoI (Takara Shuzo) and separated byagarose gel electrophoresis. Then, Southern blotting was performed todetect homologous recombinants with the probe described in Section 3,Example 48. As a result, 3 clones out of the 176 clones were homologousrecombinants. The results of Southern blot analysis of wild-type TT2Fcells and homologous recombinants #131 and #141 are shown in theleft-side three lanes in FIG. 28. In wild-type TT2F cells, two bands (aand b) are detected which were obtained by the EcoRI and XhoI digestion.In the homologous recombinants, it is expected that one of these bandsdisappears and that a new band (c) will appear at the lower part of thelane. Actually, band (a) has disappeared in #131 and #141 in FIG. 28 anda new band (c) has appeared. The size of DNA is shown at the left sideof the Figure. These results show that one allele of an antibodyheavy-chain gene in these recombinant clones has been disrupted byhomologous recombination.

EXAMPLE 50 Production of Chimeric Mice from Antibody Heavy-ChainHomologous Recombinant ES Cells

The cells in a frozen stock of the antibody heavy-chain homologousrecombinant TT2F cell clone #131 from Example 49 were thawed, started toculture and injected into 8-cell stage embryos obtained by mating a maleand a female mouse of ICR or MCH(ICR) (CREA JAPAN, INC.); the injectionrate was 10-12 cells per embryo. After the embryos were culturedovernight in the medium for ES cells (see Example 9) to develop intoblastocysts, about ten of the TT2F cell-injected embryos weretransplanted to each side of the uterus of a foster mother ICR mouse(CREA JAPAN, INC.; 2.5 days after pseudopregnant treatment). As a resultof transplantation of a total of 94 injected embryos, 22 offspring micewere born. Chimerism in the offsprings can be determined by the extentof TT2F cell-derived agouti coat color (dark brown) in the host embryo(ICR)-derived albino coat color (white). Out of the 22 offsprings, 18mice were recognized to have partial agouti coat color, indicating thecontribution of the ES cells. Out of the 18 mice, 16 mice were femalechimeric mice in which more than 80% of their coat color was agouti(i.e. ES cell-derived). From these results, it was confirmed that theantibody heavy-chain homologous recombinant ES cell clone #131 retainsthe ability to produce chimera. Since a large number of the resultantchimeric mice are female mice exhibiting extremely high contribution, itis very likely that the ES cells have differentiated into functionalgerm cells (oocytes). Two female chimeric mice exhibiting 100%contribution were mated with MCH(ICR) male mice. As a result, all of theoffspring mice exhibited agouti coat color. These offsprings are derivedfrom #131 (see Example 42), and thus it is considered that a disruptedantibody heavy-chain allele was transmitted to them at a rate of 50%.

EXAMPLE 51 Production of a Double Knockout Clone from the AntibodyHeavy-Chain Homologous Recombinant

It has been reported that a clone in which both alleles are disruptedcan be obtained by disrupting one allele by insertion of a G418resistance gene, culturing an ES cell clone in a medium with anincreased G418 concentration and screening the resultant highconcentration G418 resistant clones (Shinichi Aizawa, “Biomanual Series8, Gene Targeting”, published by Yodosha, 1995). Based on thistechnique, the inventors have conducted the following experiments inorder to obtain both alleles-disrupted clones from the TT2F antibodyheavy-chain homologous recombinants #131 and #141. First, in order todetermine the lethal concentration of G418 for both #131 and #141clones, each clone was inoculated into ten 35 mm plates at a rate ofabout 100 cells per plate (in this Example, G418 resistant primaryculture cells which were not treated with mitomycin were used as feedercells)(see Example 9). The cells were cultured in an ES mediumcontaining 0, 0.5, 1, 2, 3, 5, 8, 10, 15 and 20 mg/ml of G418(GENETICIN, Sigma) for 10 days. As a result, definite colonies wereobserved at a concentration of up to 3 mg/ml, but no colony formationwas observed at 5 mg/ml. Based on these results, the minimum lethalconcentration was decided to be 5 mg/ml. Then, high concentration G418resistant clones were selected at concentrations of 4, 5, 6, 7 and 8mg/ml. For each of #131 and #141, cells were inoculated into ten 100 mmplates at a rate of about 10⁶ cells per plate and cultured in an ESmedium containing G418 at each of the concentrations described above (5grades; two plates for each concentration). Twelve days after the startof culture, definite colonies (#131: 12 clones; #141: clones) werepicked up from plates of 7 mg/ml and 8 mg/ml in G418 concentration.These clones were stored frozen and genomic DNA was prepared by the sameprocedures as in Example 49. The genomic DNAs from these highconcentration G418 resistant clones were digested with restrictionenzymes EcoRI and XhoI (Takara Shuzo) and separated by agarose gelelectrophoresis. Then, Southern blotting was performed to detect withthe probe from Section 3, Example 48 those clones in which both alleleshave been disrupted. As a result, one clone derived from #131 (#131-3)was found to be both alleles-disrupted clone. The results of Southernblot analysis of 6 clones derived from #131 are shown in FIG. 28. Inwild-type TT2F cells, two wild-type bands (a, b) are detected after theEcoRI and XhoI digestion. In one allele homologous recombinants (#131,#141), the upper band (a) has disappeared and a new band (c) hasappeared (Example 49). Furthermore, it is expected that due to thedisruption of both alleles, another wild-type band (b) disappears andthat the disruption-type band (c) remains alone. In FIG. 28, this bandpattern is observed in clone No. 3 (#131-3). This demonstrates that bothalleles of an antibody heavy-chain gene have been disrupted in thisclone.

EXAMPLE 52 Removal of a G418 Resistance Marker Gene from the AntibodyHeavy-Chain-Deficient Homozygote TT2F Clone

The G418 resistance marker gene in the antibody heavy-chain bothalleles-disrupted clone (high concentration G418 resistant clone #131-3)from Example 51 was removed by the following procedures. An expressionvector, pBS185 (BRL), containing Cre recombinase gene which causes asite-directed recombination between the two LoxP sequences inserted atboth the ends of the G418 resistance gene was transferred into #131-3clone according to the methods described in Shinichi Aizawa, “BiomanualSeries 8, Gene Targeting”, published by Yodosha, 1995 and Seiji Takatsuet al., “Experimental Medicine (extra number): Basic Technologies inImmunological Researches”, p. 255-, published by Yodosha, 1995).Briefly, #131-3 cells were treated with trypsin and suspended in HBS togive a concentration of 2.5×10⁷ cells/ml. To the cell suspension, 30 μgof pBS185 DNA was added. Then, electroporation was performed with a genepulser (Bio-Rad Laboratories, Inc.; resistor unit not connected). Avoltage of 250 V was applied at a capacitance of 960 μF using anelectroporation cell of 4 mm in length (see Example 1). Theelectroporated cells were suspended in 5 ml of an ES medium andinoculated into a tissue culture plastic plate (Corning) of 60 mm inwhich feeder cells were seeded preliminarily. After two days, the cellswere treated with trypsin and reinoculated into three 100 mm plates(preliminarily seeded with feeder cells) such that the three plates have100, 200 and 300 cells, respectively. A similar experiment was alsoconducted under the same conditions except that the setting of the genepulser was changed (resistor unit connected; resistance value infinite).After seven days, a total of 96 colonies formed were picked up andtreated with trypsin. Then, the colonies were divided into two groups;one was inoculated into a 48-well plate preliminarily seeded with feedercells and the other was inoculated into a 48-well plate coated withgelatin alone. The latter was cultured in a medium containing 300 μg/mlof G418 (GENETICIN, Sigma) for three days. Then, G418 resistance wasjudged from the survival ratio. As a result, 6 clones died in thepresence of G418. These G418 sensitive clones were grown to confluencein 35 mm plates, and four fifths of the resultant culture was suspendedin 0.5 ml of a preservation medium [ES medium+10% DMSO (Sigma)] andstored frozen at −80° C. The remaining one fifth was inoculated into a12-well gelatin coated plate and cultured for two days. Thereafter,genomic DNA was prepared by the same procedures as in Example 2. Thesegenomic DNAs from G418 sensitive TT2F clones were digested withrestriction enzyme EcoRI (Takara Shuzo) and separated by agarose gelelectrophoresis. Then, Southern blotting was performed to confirm theremoval of the G418 resistance gene using a 3.2 kb XhoI fragment (ProbeA) from G418 resistance gene-containing pSTneoB. As a result, bandsobserved in #131-3 clone which hybridize with Probe A were not detectedat all in the sensitive clones. From these results, it was confirmedthat the G418 resistance marker gene had been surely removed in the G418sensitive clones obtained. Additionally, as a result of Southern blotanalysis performed in the same manner using Probe B obtained bydigesting pBS185 DNA with EcoRI, no specific band which hybridizes withProbe B was detected in these G418 sensitive clones. Thus, it isbelieved that Cre recombinase-containing pBS185 is not inserted into thechromosomes of the sensitive clones. In other words, these sensitiveclones can be transformed with the vector for knocking out an antibodylight-chain (vector having a loxP sequence at both the ends of a G418resistance gene) described in Section 4, Example 48.

EXAMPLE 53 Transfer of Human Chromosome #14 (Containing AntibodyHeavy-Chain Gene) into the Antibody Heavy-Chain-Deficient ES Cell Clone

Human chromosome #14 (containing an antibody heavy-chain gene) markedwith a G418 resistance gene is transferred by microcell fusion asdescribed in Example 9 into the mouse ES cell clone (from TT2F, G418sensitive) obtained in Example 52 which is deficient in an endogenousantibody heavy-chain. In the resultant G418 resistant clone, theretention of human chromosome #14 (fragment) containing a human antibodyheavy-chain gene is confirmed by PCR analysis or the like (see Example9).

EXAMPLE 54 Transfer of Human Chromosome #2 Fragment or Human Chromosome#22 into the Antibody Heavy-Chain-Deficient ES Cell Clone RetainingHuman Chromosome #14 (Fragment)

A human chromosome #2 fragment (containing the antibody heavy-chain κgene) or human chromosome #22 (containing the antibody heavy-chain λgene) marked with a puromycin resistance gene is transferred into theantibody heavy-chain-deficient mouse ES cell clone retaining a humanchromosome #14 partial fragment (G418 resistant) from Example 53 bymicrocell fusion as described in Examples 18 and 35. In the resultantpuromycin and G418 double drug-resistant clone, the retention of thehuman chromosome #14 (fragment) and human chromosome #2 fragment or #22(fragment) is confirmed by PCR analysis or the like (see Examples 18 and35).

EXAMPLE 55 Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain-Deficient Mouse ES Cells Retaining Human Chromosome #14(Fragment) Containing a Human Antibody Heavy-Chain Gene

Chimeric mice from the endogenous antibody heavy-chain gene-deficientmouse ES cell clone obtained in Example 53 retaining human chromosome#14 (fragment) containing a human antibody heavy-chain gene are producedby the same procedures as in Example 10. In the resultant chimeric mice,a human antibody heavy-chain produced in the ES cell clone-derived Bcells is detected by the method described in Example 14. Since antibodyheavy-chain genes functional in the ES cell clone-derived B cells areonly the human-derived gene on the transferred chromosome, many of theES cell clone-derived B cells produce human antibody heavy-chain.

EXAMPLE 56 Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain-Deficient Mouse ES Cells Retaining Human Chromosomes #14+#2(Fragments) or #14+#22 (Fragments)

Chimeric mice are produced by the same procedures as in Examples 19, 36,etc from the endogenous antibody heavy-chain gene-deficient mouse EScell clone retaining human chromosomes #14+#2 (fragments) or #14+#22(fragments) obtained in Example 54. In the resultant chimeric mice,human antibody heavy-chain and light-chain κ or λ are detected in the EScell clone-derived B cells according to the method described in Examples14, 23 and 32. As in Example 55, antibody heavy-chain genes functionalin the ES cell clone-derived B cells are only the human-derived gene onthe transferred chromosome. Thus, many of the ES cell clone-derived Bcells produce human heavy-chains. Furthermore, complete human antibodymolecules both heavy and light-chains which are derived from humans arealso detected by the method described in Examples 37 and 38.

EXAMPLE 57 Production of Human Antibody-Producing Hybridomas from theChimeric Mice Derived from the Endogenous Antibody Heavy-Chain-DeficientMouse ES Cells Retaining Human Chromosomes #14+#2 (Fragments) or #14+#22(Fragments)

The chimeric mice from Example 56 are immunized with an antigen ofinterest in the same manner as in Examples 15, 25 and 34. The spleen isisolated from each mice and the spleen cells are fused with myelomacells to produce hybridomas. After cultivation for 1-3 weeks, theculture supernatant is analyzed by ELISA. The ELISA is performed by themethod described in Examples 14, 15, 21, 24, 25, 33, 34, 37 and 38. As aresult, human antibody positive clones and clones which are humanantibody positive and specific to the antigen used in the immunizationare obtained.

EXAMPLE 58 Production of an Antibody Light-Chain Gene-Disrupted Clonefrom the Antibody Heavy-Chain-Deficient Homozygote Mouse ES Cells

A homologous recombinant, which has further disruption in an antibodylight-chain gene in the antibody heavy-chain-deficient homozygote TT2Fcell clone (G418 sensitive) obtained in Example 52 is produced by thefollowing procedures. Briefly, the antibody light-chain targeting vectorprepared in Section 4, Example 48 is linearized with restriction enzymeKpnI (Takara Shuzo), and transferred into the above TT2F cell clone(G418 sensitive) according to the method described in Shinichi Aizawa,“Biomanual Series 8: Gene Targeting”, published by Yodosha, 1995. After7-9 days, colonies formed are picked up. They are stored frozen andgenomic DNA is prepared in the same manner as in Example 49. GenomicDNAs from G418 resistant clones are digested with restriction enzymesEcoRI and NotI (Takara Shuzo) and separated by agarose gelelectrophoresis. Then, Southern blot analysis is performed to detecthomologous recombinants with the probe described in Section 4, Example48.

EXAMPLE 59 Production of an Double Knockout Clone from the AntibodyLight-Chain Homologous Recombinant

A clone in which both alleles of a light-chain gene are disrupted isprepared from the TT2F antibody light-chain homologous recombinant (andantibody heavy-chain-deficient homozygote) clone from Example 58 by theprocedures described below. Briefly, a high concentration G418 resistantclone is prepared and stored frozen, and DNA is prepared in the samemanner as in Example 51. Genomic DNA from the high concentration G418resistant clone is digested with restriction enzymes EcoRI and NotI(Takara Shuzo) and separated by agarose gel electrophoresis. Then,Southern blot analysis is performed to detect those clones in which bothalleles have been disrupted, with the probe from Section 4, Example 48.

EXAMPLE 60 Removal of the G418 Resistance Gene from the AntibodyLight-Chain-Deficient Homozygote (Antibody Heavy-Chain-DeficientHomozygote) TT2F Cell clone

The G418 resistance marker gene in the antibody light-chain bothalleles-disrupted clone (high concentration G418 resistant clone)obtained in Example 59 is removed by the same procedures as in Example52. Briefly, an expression vector, pBS185 (BRL), containing Crerecombinase gene which causes a site-directed recombination between thetwo loxP sequences inserted at both the ends of the G418 resistance gene(Section 1, Example 48) was transferred into the above clone accordingto the method described in Example 52. The resultant G418 sensitiveclones are grown to confluence in 35 mm plates, and 4/5 of the resultantculture was suspended in 0.5 ml of a preservation medium [ES medium+10%DMSO (Sigma)] and stored frozen at −80° C. by the same procedures as inExample 52. The remaining 1/5 was inoculated into a 12-well gelatincoated plate. After cultivation for two days, genomic DNA is prepared bythe method described in Example 2. These genomic DNAs from G418sensitive TT2F clones are digested with restriction enzyme EcoRI (TakaraShuzo) and separated by agarose gel electrophoresis. Then, Southernblotting is performed to confirm the removal of the G418 resistance geneusing a 3.2 kb XhoI fragment from G418 resistance gene-containingpSTneoB as a probe.

EXAMPLE 61 (1) Transfer of a Human Chromosome #14 Fragment (ContainingAntibody Heavy-Chain Gene) into the Endogenous Antibody Heavy-Chain andκ Chain-Deficient ES Cell Clone

A human chromosome #14 fragment SC20 (containing a human antibodyheavy-chain gene) was transferred by microcell fusion as described inSection 2 of Example 68 into the mouse ES cell clone HKD31 (from TT2F,G418 sensitive, puromycin sensitive) obtained in Example 78 which isdeficient in both endogenous antibody heavy-chain and κ chain. Themicrocell fusion and the selection of G418 resistant clones wereperformed in the same manner as in Example 2. Eight of the resultantG418 resistant clones were subjected to PCR analysis using IgM andD14S543 primers (see Example 68). As a result, both markers weredetected in 8 out of the 7 clones analyzed. Hence, it was confirmed thatthe antibody heavy-chain and κ chain-deficient ES cell clone retains thehuman chromosome #14 fragment SC20.

(2) Production of Chimeric Mice from the Endogenous Antibody Heavy-Chainand κ Chain Genes-Disrupted Mouse ES Cells Retaining a Human Chromosome#14 Fragment (Containing Antibody Heavy-Chain Gene)

Chimeric mice were produced by the same procedures as in Example 10,etc. from the endogenous antibody heavy-chain and κ chaingenes-disrupted mouse ES cell clone HKD31-8 which was obtained inSection 1 of Example 61 and which retains a human chromosome #14fragment (containing a human antibody heavy-chain gene) As a result oftransplantation of a total of 188 injected embryos, 25 offspring micewere born. Chimerism in the offsprings can be determined by the extentof TT2 cell-derived agouti coat color (dark brown) in the host embryo(ICR)-derived albino coat color (white). Out of the 25 offsprings, 17mice were recognized to have partial agouti coat color, indicating thecontribution of the ES cells. Out of the 17 mice, three were chimericmice in which more than 95% of their coat color was (ES cell-derived)agouti.

From these results, it was confirmed that the endogenous antibodyheavy-chain and κ chain genes-disrupted mouse ES cell clone retainingthe human chromosome #14 fragment (containing a human antibodyheavy-chain gene) maintains the ability to produce chimera, that is, theability to differentiate into normal tissues of mice.

(3) Detection of human antibody (having human μ, γ or α chain) in seraof the chimeric mice derived from the endogenous antibody heavy-chainand κ chain genes-disrupted mouse ES cells retaining a human chromosome#14 fragment (containing antibody heavy-chain gene)

The chimeric mice produced in Section 2 of Example 61 (derived fromHKD31-8) were bled 12 weeks (#1) or 7 weeks (#2-4) after birth. Thehuman antibody concentration in the sera was determined by ELISA in thesame manner as in Example 14. Ninety six-well microtiter plates werecoated with PBS-diluted anti-human immunoglobulin μ chain antibody(Sigma, 16385) or anti-human immunoglobulin 7 chain antibody (Sigma,13382) or anti-human immunoglobulin a chain antibody (Pharmingen,08091D) and then a serum sample diluted with mouse serum (Sigma,M5905)-containing PBS was added. Subsequently, peroxidase-labeledanti-human immunoglobulin μ chain antibody (The Binding Site Limited,MP008) or peroxidase-labeled anti-human immunoglobulin γ chain antibody(Sigma, A0170) was added to the plates and incubated. Alternatively,biotin-labeled anti-human immunoglobulin α chain antibody (Pharmingen,08092D) was added to the plates and incubated. After the plates werewashed, an avidin-peroxidase complex (Vector, ABC Kit PK4000) was addedthereto and incubated. TMBZ (Sumitomo Bakelite, ML-1120T) was added as aperoxidase substrate and then enzyme activity was determined byabsorbance measurement at 450 nm.

Purified human immunoglobulins IgM (CAPPEL, 6001-1590), IgG (Sigma,14506) and IgA (Sigma, 12636) of known concentrations having μ chain, γchain and α chain, respectively, were used as standards for determininghuman antibody concentrations in the sera. These standards were dilutedstepwise with mouse serum-supplemented PBS. The results are shown inTable 20. Chimeric mice having concentrations of human antibody μ and λchains almost as high as in normal mouse sera were confirmed. Also,chimeric mice expressing human α chain were confirmed. Further, humanimmunoglobulin γ chain sub-classes were detected in the same manner asin Example 29. As a result, all of the four subclasses (γ1, γ2, γ3 andγ4) were detected.

These results show that a human antibody heavy-chain gene is expressedefficiently in the chimeric mice derived from the endogenous antibodyheavy-chain and κ chain genes-disrupted mouse ES cells retaining thehuman chromosome #14 fragment (containing an antibody heavy-chain gene);it was also shown that not only μ chain but also all of the γ chainsubclasses and α chain were expressed therein as a result of classswitching.

TABLE 20 Human Antibody Concentrations in Chimeric Mice (ELISA) ChimericHuman Antibody (mg/l) Mouse Chimerism % IgM IgG IgA #1 90 270 1250 0.46#2 99 370 820 0.23 #3 99 550 1460 0.32 #4 95 340 2300 0.06

(4) Acquisition of Hybridomas Producing Anti-HSA Antibody ComprisingHuman γ Chain from the Chimeric Mice Derived from the EndogenousAntibody Heavy-Chain and κ Chain Genes-Disrupted Mouse ES CellsRetaining a Human Chromosome #14 Fragment (Containing AntibodyHeavy-Chain Gene)

Chimeric mice #3 (derived from HKD31-8; chimerism 99%) and #4 (chimerism95%) which had exhibited a high human antibody γ chain concentration inthe serum in Section 3 of Example 61 were immunized as described below.Human serum albumin (HSA, Sigma, A3782) dissolved in PBS was mixed withan adjuvant (MPL+TDM Emulsion, RIBI Immunochem Research Inc.) to preparean HSA solution with a concentration of 0.25 mg/ml. When theabove-described chimeric mice became 16-week old, 0.2 ml of this HSAsolution was administered intraperitoneally twice at an interval of 2weeks. Two weeks thereafter, the mice were immunized with human serumalbumin dissolved in PBS and then bled. The concentration of anti-HSAhuman antibody in the sera was determined by ELISA in the same manner asin Example 14. Briefly, ELISA plates were coated with HSA and thenperoxidase-labeled anti-human Igμ antibody (The Binding Site, MP008),anti-human Igγ antibody (Sigma, A1070) and anti-human Igα antibody(Kirkegaard & Perry Laboratories Inc., 14-10-01) were used fordetection. The results are shown FIG. 33. Hybridomas were produced usinga myeloma cell SP-2/0-Ag14 (Dainippon Pharmaceutical Co., Ltd.) by themethod described in Ando, “Monoclonal Antibody Experiment ProcedureManual”, published by Kodansha Scientific in 1991. Three days after thefinal immunization, the spleen was removed from the chimeric mice andthen cell fusion was performed using PEG in the same manner as inExample 29 to prepare hybridomas. At the same time, blood samples werecollected from the mice to quantitate human Igγ subclasses in the sera.As a result, 920 mg/l of γ1, 520 mg/l of γ2, 11 mg/l of γ3 and 140 mg/lof γ4 were detected in the serum of chimeric mouse #3.

The fused cells were diluted with a medium (Sanko Pure Chemical, SCloning Medium CM-B) containing 5% HCF (Air Brown) and HAT (DainipponPharmaceutical Co., Ltd., No. 16-808-49) or 1 mg/ml of G418 to give aconcentration of 10⁶ spleen cells/ml and then dispensed into 96-wellplates (100 μl/well), followed by cultivation. At day 8 of thecultivation, the culture supernatant was collected and screened forhuman antibody-producing hybridomas by ELISA in the same manner as inExample 14. Briefly, ELISA plates were coated with a HSA solutiondissolved in CBB buffer to give a concentration of 5 μg/ml.Peroxidase-labeled anti-human immunoglobulin γ chain antibody (Sigma,A0170) and TMBZ (Sumitomo Bakelite, ML-1120T) were used for detection.An absorbance about 3 times higher than the absorbance in the negativecontrol was used as a criterion for judgement. As a result, 74 positivewells were obtained from chimeric mouse #3 and 29 positive wells fromchimeric mouse #4. Also, anti-HSA antibody having human μ chain wasscreened in HSA-solution-coated plates using peroxidase-labeledanti-human immunoglobulin A chain antibody (Tago, #2392). Briefly, fusedcells from chimeric mouse #3 were inoculated into fifteen 96-wellplates, from which 4 plates were selected by G418 resistance. Theculture supernatants of these 4 plates were screened to obtain 5positive wells. Wells which exhibited colony formation after selectionwith HAT or 1 mg/ml of G418 were 74 wells/plate for HAT and 29wells/plate for G418. The cells of those wells which were positive forhuman γ chain-containing anti-HSA antibody and which had a relativelylarge number of cells were transferred into 46-well plates and culturedfor another 4 days. The isotype of the antibody in the culturesupernatant was determined by ELISA. ELISA was performed in HSA-coatedplates using alkali phosphatase-labeled anti-human IgG1 antibody (ZymedLabolatories, Inc., 05-3322), anti-human IgG2 antibody (ZymedLabolatories, Inc., 05-3522), anti-human IgG3 antibody (ZymedLabolatories, Inc., 05-3622) and anti-human IgG4 antibody (ZymedLabolatories, Inc., 05-3822) in the same manner as in Example 14. As aresult, 27 human IgG1 positive clones, 11 human IgG2 positive clones, 2human IgG3 positive clones and 13 human IgG4 positive clones wereobtained. Fused cells from chimeric mouse #4 were treated in the samemanner to obtain 4 positive clones with a large number of cells as humanIgG1 producing clones.

These results show that the immunization by human protein (HSA) of thechimeric mice derived from the endogenous antibody heavy-chain &light-chain-deficient mouse ES cells retaining the human chromosome #14partial fragment containing a human antibody heavy-chain gene increasesthe antibody titers of antigen specific human Igμ, γ and α to therebyenable the acquisition of hybridomas producing anti-HSA antibodycontaining μ chain and all of the human γ chain subclasses.

EXAMPLE 62 Transfer of Human Chromosome #2 (Containing Light-Chain κGene) into the Endogenous Antibody Heavy-Chain and κ Chain-Deficient ESCells Retaining a Human Chromosome #14 Fragment (Containing AntibodyHeavy-Chain Gene)

A human chromosome #2 fragment (containing antibody light-chain κ gene)marked with a puromycin resistance gene was transferred into theendogenous antibody heavy-chain and κ chain-deficient mouse ES cellclone HKD31-8 obtained in Section 1 of Example 61 and which retained ahuman chromosome #14 fragment (containing an antibody heavy-chain gene).The method of transfer was by microcell fusion as described in Example18. As a result, 13 puromycin and G418 double-resistant clones wereobtained. These clones were subjected to PCR analysis (see Example 18)using IgM and D14S543 primers (see Example 68) for the chromosome #14fragment and V κ1 and FABP1 primers (see Example 12) for the chromosome#2 fragment. As a result, the presence of all the 4 markers wasconfirmed in 8 clones. Of these clones, KH13 clone was subjected to FISHanalysis using human chromosome-specific probes (see Examples 9 and 12).The results are shown in FIG. 34. Two independent, small chromosomefragments hybridizing to the probes were observed in KH13. These resultsshow that KH13 retains both the chromosome #14 fragment and thechromosome #2 fragment.

EXAMPLE 63 Transfer of Human Chromosome #22 (Containing Light-Chain λGene) into the Endogenous Antibody Heavy-Chain and κ Chain-Deficient ESCells Retaining a Human Chromosome #14 Fragment (Containing AntibodyHeavy-Chain Gene)

Human chromosome #22 (containing antibody light-chain λ gene) markedwith a puromycin resistance gene was transferred into mouse ES cellclone HKD31-8 obtained in Section 1 of Example 61 which was deficient inthe endogenous antibody heavy-chain & κ chain and which retained a humanchromosome #14 fragment (containing an antibody heavy-chain gene). Themethod of transfer was by microcell fusion as described in Example 35.As a result, 12 puromycin and G418 double drug-resistant clones wereobtained. These clones were subjected to PCR analysis (see Example 35)using IgM and D14S543 primers for the chromosome #14 fragment and Igλ,D22S315, D22S275, D22S278, D22S272 and D22S274 primers (see Example 2)for the chromosome #22 fragment. As a result, the presence of all of the8 markers was confirmed in 10 clones. Of the remaining 2 clones, LH13clone exhibited the presence of 5 markers, IgM, D14S543, IgI, D22S275and D22S274. Thus, it is believed that this clone contains a fragment ofhuman chromosome #22. LH13 was further subjected to FISH analysis usinga human chromosome #22-specific probe and a human chromosome#14-specific probe separately. As a result, independent chromosomefragments hybridizing to the respective probes were observed. Thisindicates that this clone retains both a chromosome #14 fragment and achromosome #2 fragment.

EXAMPLE 64 Production of Endogenous Antibody Heavy-Chain &Light-Chain-Deficient Mouse ES Cells Retaining Three Human Chromosomes,#2 (Containing Antibody Light-Chain κ Gene), #14 (Containing AntibodyHeavy-Chain Gene) and #22 (Containing Antibody λ Chain Gene), or PartialFragments Thereof

In order to obtain mouse ES cells retaining three kinds of humanchromosomes, human chromosome #2 or #22 is marked by inserting a markergene such as blasticidin resistance (Izumi et al., Exp. Cell. Res.,197:229, 1991), hygromycin resistance (Wind et al., Cell, 82:321-,1995), etc. This marking is performed according to the method describedin Examples 16 and 26. Human chromosome #22 (containing human antibodylight-chain λ gene) marked with blasticidin resistance, hygromycinresistance, etc. is transferred into the mouse ES cell clone (from TT2F,G418 resistant, puromycin resistant) obtained in Example 62 which isdeficient in endogenous antibody heavy-chain & light-chain and whichretains both human chromosome #14 (fragment) and human chromosome #2(partial fragment). The method of transfer is by the method described inExample 9. As feeder cells for culturing ES cells, appropriate cells areselected depending on the selection marker used. When a hygromycinresistance marker is used, primary culture fibroblasts obtained from atransgenic mouse strain which retains and expresses the marker (Johnsonet al., Nucleic Acids Research, vol. 23, No. 7, 1273-, 1995) are used.It is confirmed by PCR analysis, etc. (see Examples 9, 18 and 35) thatthe resultant G418, puromycin and hygromycin (or blasticidin) tripledrug-resistant clones retain the three kinds of human chromosomes(fragments) described above. In the same manner, a human chromosome #2fragment marked with a hygromycin or blasticidin resistance gene istransferred into the mouse ES cell clone (from TT2F, G418 resistant,puromycin resistant) obtained in Example 63 which is deficient inendogenous antibody heavy-chain & light-chain and which retains bothhuman chromosome #14 (fragment) and human chromosome #22 (fragment).

EXAMPLE 65 Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain & Light-Chain Genes-Disrupted Mouse ES Cells Retaining aPlurality of Human Chromosomes (Fragments) Containing Human AntibodyHeavy-Chain Gene and Light-Chain Gene, Respectively

Chimeric mice are produced by the same procedures as in Example 10, etc.from the endogenous antibody heavy-chain & light-chain genes-disruptedmouse ES cell clones that retain human chromosomes (fragments)containing human antibody genes and which were obtained in Examples 61,62, 63 and 64. In the resultant chimeric mice, mouse antibodies producedin host embryo-derived B cells and human antibodies produced mainly inES cell clone-derived B cells are detected by the method described inExamples 14, 23 and 32. Since the antibody heavy-chain gene and thelight-chain κ gene which are both functional in the ES cellclone-derived B cells are only human-derived genes on the transferredchromosomes, many of the ES cell clone-derived B cells produce humanantibody heavy-chain and light-chain κ (Lonberg et al., Nature,368:856-, 1994). Furthermore, complete human antibody molecules in whichboth heavy- and light-chains are derived from human are also detected bythe method described in Examples 37 and 38.

(1) Production of Chimeric Mice from the Endogenous Antibody Heavy-Chainand κ Chain Genes-Disrupted Mouse ES Cells Retaining Both a HumanChromosome #14 Fragment (Containing Antibody Heavy-Chain Gene) and aHuman Chromosome #2 Fragment (Containing Antibody Light-Chain κ Gene)

Chimeric mice were produced by the same procedures as in Example 10,etc. from mouse ES cell clone KH13 obtained in Example 62 which isdeficient in the endogenous antibody heavy-chain and κ chain genes andwhich retains both a human chromosome #14 fragment (containing anantibody heavy-chain gene) and a human chromosome #2 fragment(containing light-chain κ gene) As a result of transplantation of atotal of 176 injected embryos, 20 offspring mice were born. Chimerism inthe offsprings can be determined by the extent of TT2 cell-derivedagouti coat color (dark brown) in the host embryo (ICR)-derived albinocoat color (white). Out of the 20 offsprings, 7 mice were recognized tohave partial agouti coat color, indicating the contribution of the EScells.

From these results, it was confirmed that the endogenous antibodyheavy-chain and κ chain genes-disrupted mouse ES cell clone retainingboth a human chromosome #14 fragment (containing an antibody heavy-chaingene) and a human chromosome #2 fragment (containing light-chain κ gene)maintains the ability to produce chimera, that is, the ability todifferentiate into normal tissues of mice.

(2) Production of Chimeric Mice from the Endogenous Antibody Heavy-Chainand κ Chain Genes-Disrupted Mouse ES Cells Retaining Both a HumanChromosome #14 Fragment (Containing Antibody Heavy-Chain Gene) and aHuman Chromosome #22 Fragment (Containing Light-Chain λ Gene)

Chimeric mice were produced by the same procedures as in Example 10,etc. from mouse ES cell clone LH13 obtained in Example 63 which isdeficient in the endogenous antibody heavy-chain and κ chain genes andwhich retains both a human chromosome #14 fragment (containing anantibody heavy-chain gene) and a human chromosome #22 fragment(containing light-chain λ gene). As a result of transplantation of atotal of 114 injected embryos, 22 offspring mice were born. Chimerism inthe offsprings can be determined by the extent of TT2 cell-derivedagouti coat color (dark brown) in the host embryo (ICR)-derived albinocoat color (white). Out of the 22 offsprings, 5 mice were recognized tohave partial agouti coat color, indicating the contribution of the EScells.

From these results, it was confirmed that the endogenous antibodyheavy-chain and κ chain genes-disrupted mouse ES cell clone retainingboth a human chromosome #14 fragment (containing an antibody heavy-chaingene) and a human chromosome #22 fragment (containing light-chain λgene) maintains the ability to produce chimera, that is, the ability todifferentiate into normal tissues of mice.

(3) Detection and Quantitative Determination of Complete Human Antibodyin Sera of the Chimeric Mice Derived from the Endogenous AntibodyHeavy-Chain and κ Chain-Deficient Mouse ES Cells Retaining Both a HumanChromosome #2 Partial Fragment and a Human Chromosome #14 PartialFragment

The chimeric mice (derived from KH13) produced in Section 1 of Example65 were bled at day 40 after birth. The concentrations of human antibodyin the sera were determined by ELISA in the same manner as in Example14. Briefly, ELISA plates were coated with PBS-diluted anti-humanimmunoglobulin κ chain antibody (Kirkegaard & Perry Labolatories Inc.,01-10-10) or anti-human immunoglobulin κ chain antibody (Vector,AI-3060) and then serum samples diluted with mouse serum (Sigma,M5905)-supplemented PBS were added. Subsequently, peroxidase-labeledanti-human immunoglobulin μ chain antibody (The Binding Site Limited,MP008) or peroxidase-labeled anti-human immunoglobulin γ chain antibody(Sigma, A0170) was added and incubated. TMBZ (Sumitomo Bakelite,ML-1120T) was added as a peroxidase substrate and then enzyme activitywas determined by absorbance measurement at 450 nm. Purified humanimmunoglobulins IgM (Caltag, 13000) and IgG (Sigma, I4506) of knownconcentrations having μ chain and κ chain were used as standards fordetermining human antibody concentrations in the sera by comparison.These standards were diluted stepwise with mouse serum-supplemented PBS.The results are shown in Table 21. Chimeric mice were confirmed that hadconcentrations of complete human antibody more than 10 times higher thanin chimeric mice derived from ES cells whose endogenous antibody geneswere not knocked out. Also, complete human antibody containing human γchain was confirmed in the sera of the chimeric mice.

From these results, it was confirmed that the concentration of completehuman antibody in which both heavy- and light-chains were derived fromhuman increased in the chimeric mice derived from the endogenousantibody heavy-chain and κ chain-deficient mouse ES cells retaining botha human chromosome #14 partial fragment and a human chromosome #22partial fragment.

TABLE 21 Concentrations of Human Antibodies in Chimeric Mice (ELISA)Chimeric ES clone mouse Chimerism (%) IgM, κ (mg/l) IgG, κ (mg/l) KH13CKH13-1 95 0.1 0.07 KH13 CKH13-2 85 0.9 0.13(4) Detection and Quantitative Determination of Complete Human Antibodyin Sera of the Chimeric Mice Derived from the Endogenous AntibodyHeavy-Chain and κ Chain-Deficient Mouse ES Cells Retaining Both a HumanChromosome #14 Partial Fragment and a Human Chromosome #22 PartialFragment

The chimeric mice (derived from KH13) produced in Section 2 of Example65 were bled at day 49 after birth. The concentrations of human antibodyin the sera were determined by ELISA in the same manner as in Example14. Briefly, ELISA plates were coated with PBS-diluted anti-humanimmunoglobulin λ chain antibody (Kirkegaard & Perry Labolatories Inc.,01-10-11) or anti-human immunoglobulin λ chain antibody (Vector,AI-3070) and then serum samples diluted with mouse serum (Sigma,M5905)-supplemented PBS were added. Subsequently, peroxidase-labeledanti-human immunoglobulin μ chain antibody (The Binding Site Limited,MP008) or peroxidase-labeled anti-human immunoglobulin γ chain antibody(Sigma, A0170) was added and incubated. TMBZ (Sumitomo Bakelite,ML-1120T) was added as a peroxidase substrate and then enzyme activitywas determined by absorbance measurement at 450 nm. Purified humanimmunoglobulins IgM (Caltag, 13000) and IgG (Sigma, I4506) of knownconcentrations having μ chain and κ chain were used as standards fordetermining human antibody concentrations in the sera by comparison.These standards were diluted stepwise with mouse serum-supplemented PBS.The results are shown in Table 22. Chimeric mice individuals wereconfirmed that had concentrations of complete human antibody about 40times higher than in chimeric mice derived from ES cells whoseendogenous antibody genes were not knocked out. Also, complete humanantibody containing human γ chain was confirmed in the sera of thechimeric mice.

From these results, it was confirmed that the concentration of completehuman antibody in which both heavy- and light-chains were derived fromhuman increased in the chimeric mice derived from the endogenousantibody heavy-chain and κ chain-deficient mouse ES cells retaining botha human chromosome #14 partial fragment and a human chromosome #22partial fragment.

TABLE 22 Concentrations of Human Antibodies in Chimeric Mice (ELISA)Chimeric ES clone mouse Chimerism (%) IgM, λ (mg/l) IgG, λ (mg/l) LH13CLH13-1 95 13 2.6 LH13 CLH13-2 90 2.8 0.36

EXAMPLE 66 Production of Complete Human Antibody-Producing Hybridomasfrom Chimeric Mice Prepared by Transferring the Endogenous AntibodyHeavy-Chain and Light-Chain-Deficient Mouse ES Cells Retaining Both aHuman Chromosome #14 Partial Fragment and a Human Chromosome #22 PartialFragment into Immunodeficient Mouse Host Embryos

A chimeric mouse CLH13-3 (derived from TT2FES clone LH13; chimerism 35%)obtained in Section 3 of Example 67 was immunized with HSA from day 43after birth. Briefly, human serum albumin (HSA, Sigma, A3782) dissolvedin PBS was mixed with an adjuvant (MPL+TDM Emulsion, RIBI ImmunochemResearch Inc.) to prepare a HSA solution with a concentration of 0.25mg/ml, 0.2 ml of which was administered intraperitoneally twice at aninterval of 1 week. One week thereafter, the mouse was immunized withhuman serum albumin dissolved in PBS. The mouse was bled every 1 week todetermine the concentrations of anti-HSA human antibodies in the serumby ELISA in the same manner as in Example 14. The results are shown inFIG. 35. The spleen was removed from the chimeric mouse at day 3 afterthe final immunization and then cell fusion was performed using PEG inthe same manner as in Example 24 to prepare hybridomas. Briefly, thefused cells were diluted with a medium (Sanko Pure Chemical, S CloningMedium CM-B) containing HAT (Dainippon Pharmaceutical Co., Ltd., No.16-808-49) or 1 mg/ml of G418 to give a concentration of 10⁶ spleencells/ml and then dispensed into 96-well plates (100 μl/well), followedby cultivation. Both of the selection media contained 5% HCF (AirBrown). At day 6 of the cultivation, colonies were formed in almost allwells in both the G418 selection and HAT selection plates. A total ofabout 770 hybridoma-positive wells were obtained. The culturesupernatants were collected and subjected to screening for humanantibody-producing hybridomas by ELISA in the same manner as in Example14. Briefly, ELISA plates were coated with anti-human immunoglobulin λchain antibody (Vector, AI-3070) Biotin-labeled anti-humanimmunoglobulin λ chain antibody (Vector, BA-3070) and anavidin-peroxidase complex (Vector ABC Kit PK4000) were used fordetection with TMBZ (Sumitomo Bakelite, ML-1120T) used as a substrate.An absorbance about 2 times higher than the absorbance in the negativecontrol was used as a criterion for judgement. As a result, 17 positivewells were obtained. The cells of the positive wells were transferredinto 24-well plates and cultured in IMDM medium containing 10% FBS. Theculture supernatants were analyzed by ELISA in the same manner as inSection 4 of Example 65. As a result, the presence of 0.09-11 mg/ml ofcomplete human antibody having both human Igμ & Igλ was confirmed in 16wells. The antibody titer of anti-HSA human λ chain was determined inthe same manner as in Example 33 to obtain one positive well. The cellsof the well which was complete human antibody-positive and anti-HSAhuman λ chain-positive were cloned by limiting dilution according to themethod described in Ando, “Monoclonal Antibody Experiment ProcedureManual”, published by Kodansha Scientific in 1991. As a result, 2 clonesof anti-HSA human λ chain-positive hybridomas were obtained.

From these results, it was confirmed that complete humanantibody-producing hybridomas could be obtained from chimeric miceprepared by transferring the endogenous antibody heavy-chain andlight-chain-deficient mouse ES cells retaining both a human chromosome#14 partial fragment and a human chromosome #22 partial fragment intoimmunodeficient mouse host embryos. Furthermore, it was confirmed thatthe antibody titers of antigen-specific human Igμ and Igλ increased inresponse to the stimulation with the HSA antigen. It was furtherconfirmed that hybridomas producing a HAS-specific antibody consistingof human Igμ and Igλ could be obtained from this chimeric mouse.

Since the fused cells had a drug resistance marker on their chromosome,it was possible to select hybridomas using G418 without adding HAT.After G418 selection, only those cells having a human chromosome growand, thus, hybridomas can be obtained selectively. Also, it is expectedthat a human chromosome can be prevented from falling off fused cells.Furthermore, it is expected that even myeloma cells unsuitable for HATselection such as those having HGPRT(hypoxanthine-guanine-phosphoribosyltransferase) enzyme may becomeavailable for cell fusion.

EXAMPLE 67 Production of Chimeric Mice with Heavy-Chain Gene-DisruptedHost Embryos

From those mice exhibiting agouti coat color among the progeny of theendogenous antibody heavy-chain one allele-disrupted TT2F cellclone-derived chimeric mice produced in Example 49, mice retaining thedisrupted allele are selected by Southern blot analysis (Example 49) orthe like (the expected possibility is 1/2). Offsprings born by themating of those antibody heavy-chain-deficient heterozygous male andfemale mice are subjected to Southern blot analysis (see Example 49),analysis of the production of antibody heavy-chains in sera (Kitamura etal., Nature, 350:423-, 1991), etc. Thus, antibody heavy-chain-deficienthomozygotes can be obtained which are deficient in both alleles andwhich can hardly produce functional antibodies of their own (theexpected possibility is 1/4; for the results in membrane-type μchain-deficient mice, see Kitamura et al., Nature, 350:423-, 1991).

(1) Establishment of an Antibody Heavy-Chain Knockout Mouse Strain

Those mice that exhibited agouti coat color among the progeny of theendogenous antibody heavy-chain one allele-disrupted TT2F cellclone-derived chimeric mice produced in Example 49 were subjected toSouthern blot analysis (Example 49) to select those mice that retainedthe disrupted allele. Offsprings born by the mating of these antibodyheavy-chain-deficient heterozygous male and female mice were subjectedto Southern blot analysis (see Example 49) and analysis of theproduction of antibody μ chain in sera (Kitamura et al., Nature,350:423-, 1991), etc. As a result, antibody heavy-chain-deficienthomozygotes could be obtained which were deficient in both alleles andwhich could hardly produce functional antibodies of their own (for theresults in membrane-type μ chain-deficient mice, see Kitamura et al.,Nature, 350:423-, 1991).

Thus, an antibody heavy-chain knockout mouse strain could be establishedfrom the antibody heavy-chain one allele-disrupted TT2F cell clone.

Embryos obtained by mating the homozygous male and female mice bred in aclean environment may be used as hosts for producing chimeric mice. Inthis case, most of the B cells functional in the resultant chimeric miceare derived from the injected ES cells. Other mouse strains which cannotproduce their own functional B cells, such as RAG-2-deficient mouse(Sinkai et al., Cell, 68:855-, 1992), may also be used for this purpose.In this system, chimeric mice are produced by the same procedures as inExample 10, etc. using the mouse ES cell clone from Examples 62, 63 or64 which is deficient in endogenous antibody heavy-chain & light-chainand which retains human chromosomes #14+#2, #14+#22 or #14+#2+#22(fragments). The resultant chimeric mice mainly produce human antibodiesby the expression of human antibody heavy-chain (on chromosome #14),light-chain κ (on chromosome #2) and light-chain λ (on chromosome #22)genes that are functional in ES cell-derived B cells.

(2) Detection and Quantitative Determination of Complete Human Antibodyin Sera of the Chimeric Mice Produced by Injecting the EndogenousAntibody Heavy-Chain and κ Chain-Deficient Mouse ES Cells Retaining botha Human Chromosome #2 Partial Fragment and a Human Chromosome #14Partial Fragment into Immunodeficient Mouse Host Embryos

Chimeric mice were produced in the same manner as in Section 1 ofExample 65 by injecting the ES cell clone KH10 from Example 62 into theembryos obtained by mating male and female mice of the antibodyheavy-chain knockout mouse strain established in Section 1 of Example67. Seven-week old resultant chimeric mice were bled to determine theconcentrations of human antibodies in the sera by ELISA in the samemanner as in Example 14 and Section 3 of Example 65. The results areshown in Table 23. Complete human antibodies having human μ chain+κchain and human γ chain+κ chain, respectively, were confirmed in thesera of the chimeric mice. It was also confirmed that by transferring EScells into immunodeficient host embryos, complete antibodies could beobtained even in the resultant chimeric mice of low chimerism since Bcells are differentiated only from ES cells.

TABLE 23 Concentrations of Human Antibodies in Chimeric Mice (ELISA)Chimeric ES clone mouse Chimerism (%) IgM, κ (mg/l) IgG, κ (mg/l) KH13CKH10-1 6 6.1 0.17 KH13 CKH10-2 3 1.9 0.4(3) Detection and Quantitative Determination of Complete Human Antibodyin Sera of the Chimeric Mice Produced by Injecting the EndogenousAntibody Heavy-Chain and κ Chain-Deficient Mouse ES Cells Retaining botha Human Chromosome #14 Partial Fragment and a Human Chromosome #22Partial Fragment into Immunodeficient Mouse Host Embryos

Chimeric mice were produced in the same manner as in Section 2 ofExample 65 by injecting the ES cell clone LH13 from Example 62 into theembryos obtained by mating male and female mice of the antibodyheavy-chain knockout mouse strain established in Section 1 of Example67. Five-week old resultant chimeric mice were bled to determine theconcentrations of human antibodies in the sera by ELISA in the samemanner as in Example 14 and Section 4 of Example 65. The results areshown in Table 24. Complete human antibodies having human μ chain+chainand human γ chain+λ chain, respectively, were confirmed in the sera ofthe chimeric mice.

TABLE 24 Concentrations of Human Antibodies in Chimeric Mice (ELISA) ESclone Chimeric mouse Chimerism (%) IgM, λ (mg/l) LH13 CLH13-3 35 51 LH13CLH13-4 85 32 LH13 CLH13-4 30 27

EXAMPLE 68 Retention of the Human Chromosome in Offsprings of HumanChromosome #14 Fragment (Containing Antibody Heavy-ChainGene)-Transferred ES Cell-Derived Chimeric Mice (1) Isolation ofHuman-Mouse Hybrid Cells Retaining a Human Chromosome #14 FragmentContaining an Antibody Heavy Chain Gene

It was observed in Example 42 that a human chromosome #2 fragmenttransferred into mice was transmitted to their progeny. Thus, it isexpected that the possibility of transmission of human chromosome #14 toprogeny will be increased if a fragment of this chromosome is used.A9/#14 clone (Example 9; corresponding to A9/14-C11 clone described inTomizuka et al., Nature Genet. vol 16, 133-143 (1997)) retaining anintact human chromosome #14 marked with a G418 resistance gene wassubjected to a more detailed FISH analysis (Example 9). As a result, itwas observed that about 10% of the cell population contained only a verysmall, fragmented human chromosome #14. This chromosome fragment isalmost of the same size as the chromosome #2 fragment (Example 12) andbelieved to contain the G418 resistance marker.

In order to isolate the cell clones containing the fragmented humanchromosome #14, (about 300) A9/#14 cells were seeded on 10 cm plates andcultured. At day 10 of the cultivation, 31 colonies were picked up.Genomic DNAs were prepared from these clones and subjected to PCRanalysis in the same manner as in Example 9 using chromosome #14specific primers (the 18 primers shown in Example 9 were used except PCIand NP). Out of the 16 primers, only IgM, IGG1, IGA2 and IGVH3 werefound in one clone (A9/SC20). Since D14S543 (Science, HUMAN GENETIC MAP(1994); the base sequence was obtained from databases of GenBank, etc.)which is a marker located near the human chromosome #14 long armtelomere was also detected in the clone, the fragment of interest(hereinafter referred to as “SC20 fragment”) retained in the clone isbelieved to contain a region adjacent to the chromosome #14 telomere andwhich contained an antibody heavy-chain gene.

SC20 fragment was subjected to FISH analysis (Tomizuka et al., NatureGenet. vol 16, 133-143 (1997)) using a human chromosome-specific probe.As a result, it was observed that the size of the chromosome in theclone that hybridized to the probe was smaller than in the control clone(containing an intact chromosome #14). Thus, it was confirmed thatA9/SC20 contained a fragment of human chromosome #14.

Further, in order to examine whether SC20 fragment contained a humanchromosome #14-derived centromere sequence, chromosome samples fromA9/SC20 cells were hybridized to digoxigenin-11-dUTP-labeled humanchromosome #14 or #22-specific α satelite DNA (purchased from COSMOBIO)which was used as a probe, followed by FISH analysis according to themethod described in a reference (Tomizuka et al., Nature Genet. vol 16,133-143 (1997)). As a result, a signal hybridizing to the above probewas confirmed. Thus, it has become clear that SC20 fragment contains ahuman-derived centromere sequence (FIG. 36).

(2) Transfer of Human Chromosome #14 (Fragment) into TT2F Cells andStable Retention of the Chromosome Therein

A human chromosome #14 fragment was microcell-transferred into TT2Fcells using A9/SC20 as a chromosome donor cell in the same manner as inExample 9. As a result of G418 (300 μg/ml) selection, 5 resistant cloneswere obtained. These ES cell clones were subjected to PCR analysis(Example 68, Section 1) and FISH analysis (using humanchromosome-specific probes; Tomizuka et al., supra) to confirm theretention of a human chromosome #14 fragment. The results of the FISHanalysis are shown in FIG. 37.

Stable retention of transferred human chromosomes in mice is importantfor efficient expression of the transferred genes and efficienttransmission of the transferred chromosomes to their progeny. Sinceselection by addition of drugs is impossible after an ES cell clone hasbeen injected into host embryos, it is desired that transferred humanchromosomes be retained stably even under non-selective conditions.

TT2F(SC20)-21 clone containing SC20 fragment was cultured in a mediumnot containing G418 for a long period to examine the retention of SC20fragment under this condition.

Briefly, TT2F(SC20)-21 clone was cultured in a selective medium (G418:300 μg/ml) for 1 week and then subcultured in a non-selective medium for45 days (subcultured every other day with 8-fold dilution). At day 0,15, 30 and 45 of the subculture, 300-1000 cells were seeded in six 35 mmplates, three of which contained the selective medium and the otherthree non-selective medium. The cells were cultured in these plates forabout 1 week and then the colonies were counted. Chromosome retentionratio (A/B) was calculated by dividing the total number of colonies inthe 3 plates under selective conditions (A) by the total number ofcolonies in the 3 plates under non-selective conditions (B). For thepurpose of comparison, an experiment was conducted using P-21 clone(Example 40) containing W23 fragment derived from human chromosome #2 inthe same manner as described above (the selective medium contained 0.75μg/ml of puromycin). The results are shown in FIG. 38. The values shownin FIG. 38 are average values from 3 independent experiments. SC20fragment exhibited a high retention ratio of 95% or above even after the45 day cultivation under non-selective conditions. On the other hand,W23 fragment exhibited a retention ratio of 14% under identicalconditions.

There have been reports of the transfer of a human Y chromosome-derivedartificial chromosome (containing human Y-derived centromere) into CHO(hamster fibroblasts), DT40 (chicken B lymphocytes) and mouse ES cell(Shen et al., Hum. Mol. Genet. 6, 1375-1382). Under non-selectivecultivation, the artificial chromosome was retained stably in CHO andDT40. In mouse ES cells, however, only the chromosome which accidentallyacquired the mouse centromere as a result of rearrangement was retainedstably. From these results, an opinion was proposed that a human-derivedcentromere is unstable in mouse ES cells (Shen et al., supra)

The results described above show that SC20, though it contains ahuman-derived centromere (Example 68, Section 1), is very stable inmouse ES cells. Since the retention of W23 fragment (which is alsosuggested to contain a human-derived centromere) (Tomizuka et al.,supra) in mouse ES cells appeared to be unstable, it is considered thatthe stability of human-derived chromosomes in mouse ES cells variesdepending on the type of the chromosome.

From these results, it was demonstrated that SC20 fragment is veryuseful as a vector for transferring a gene into mice.

(3) Production of Chimeric Mice from the ES Cell Clone Retaining HumanChromosome #14 (Fragment)

Cells in the frozen stock of G418 resistant ES cell clone TT2F (SC20)-21which was obtained in Example 68, Section 2 and which was confirmed toretain a human chromosome #14 fragment were thawed, started up forculture and injected into 8-cell stage embryos obtained by mating maleand female mice of ICR (CREA JAPAN, INC.); the injection rate was 10-12cells per embryo. After the embryos were cultured overnight in themedium for ES cells (see Example 9) to develop into blastocysts, about10 of the injected embryos were transplanted to each side of the uterusof foster mother ICR mice (CREA JAPAN, INC.; 2.5 days afterpseudopregnant treatment).

As a result of transplantation of a total of 188 injected embryos, 22offspring mice were born. Chimerism in the offsprings can be determinedby the extent of TT2 cell-derived agouti coat color (dark brown) in thehost embryo (ICR)-derived albino coat color (white). Out of the 22offsprings, 20 mice were recognized to have partial agouti coat color,indicating the contribution of the ES cells. Out of the 20 mice, twowere chimeric mice in which their coat color was complete agouti (i.e.ES cell-derived).

From these results, it was confirmed that ES cell clone TT2F(SC20)-21retaining a human chromosome #2 fragment maintains the ability toproduce chimera, that is, the ability to differentiate into normaltissues of mice.

Two 5-week old chimeric mice [derived from TT2F(SC20)-21, chimerism100%, C14m-16 and -17] were bled to determine the concentrations ofhuman antibody IgM and IgG in the sera by ELISA in the same manner as inExample 14. The results are shown in Table 25.

TABLE 25 Concentrations of Human Antibody Heavy- Chains in Chimeric Mice(ELISA) Chimeric mouse Chimerism (%) IgM (mg/l) IgG (mg/l) C14m-16 1007.9 1.0 C14m-17 100 6.0 1.3

Human antibodies IgM and IgG were detected in the sera of both chimericmice. The concentrations of these human antibodies were comparable tothe concentrations in the chimeric mice retaining the larger humanchromosome #14 fragment (see Example 14). Thus, it was demonstrated thatthe human antibody gene contained in SC20 fragment is functional.

(4) Confirmation of the Retention of Human Chromosome in the Progeny ofChimeric Mice Derived from the Mouse ES Cells (TT2F, XO) Retaining aHuman Chromosome #14 Fragment, and Detection and QuantitativeDetermination of Human Antibody μ Chain and γ Chain in Sera of theProgeny

Examination was Made as to Whether ES Cell-Derived offsprings would beproduced by mating the female chimeric mice C14m-16 and C14m-17 (bothhaving 100% chimerism in coat color) from Example 68, Section 3 withmale ICR mice. By this mating, offsprings with agouti coat color shouldbe produced from TT2F cell (agouti: dominant)-derived oocytes in thechimeric mice fertilized by male ICR mouse (albino: recessive)-derivedsperms, and offsprings with albino coat color should be produced fromICR-derived oocytes in the chimeric mice. Actually, all of the viableoffspring mice obtained by this mating (30 in total) exhibited EScell-derived agouti coat color, indicating efficient transmission of EScells to the germ cell lineage. Genomic DNAs were prepared from thetails of these offspring mice to examine the retention of a humanchromosome fragment by PCR. PCR amplification was performed using thethree primers (IGVH3, IgM and D14S543) of which the presence inTT2F(SC20)-21 was confirmed. As a result, the presence of the threemarkers detected in TT2F (SC20)-21 was confirmed in 10 out of the 30offspring mice (33%). These results show that TT2F cell cloneTT2F(SC20)-21 retaining a human chromosome #14 fragment differentiatedinto functional oocytes in the chimeric mice and that the humanchromosome #14 fragment was transmitted to the F1 progeny derived fromthe oocytes.

Detection and quantitative determination of human antibodies IgM and IgGin sera were performed on 9 out of the 10 offspring mice which wereconfirmed to retain a human chromosome #14 fragment, as described below.About 4-8 week-old mice were bled to detect human antibody μ chain and γchain by ELISA in the same manner as in Example 14. As a result, humanantibody μ chain and γ chain were detected in the sera of all of themice tested (see Table 26). Thus, It was confirmed that the humanantibody heavy chain gene also functions in the F1 progeny born by thechimeric mice.

TABLE 26 Concentrations of Human Antibodies IgM and IgG in Chimeric Mice(ELISA) Mother Mouse IgG Chimeric Mouse Individual No. IgM (mg/l) (mg/l)C14m-16 16-5  12.9 2.2 C14m-16 16-14 3.5 2.2 C14m-16 16-16 4.1 2.0C14m-16 16-17 5.5 3.9 C14m-17 17-7  5.7 1.0 C14m-17 17-8  3.6 1.2C14m-17 17-19 3.5 0.75 C14m-17 17-22 2.4 1.4 C14m-17 17-23 5.3 1.9

Further, 3 male mice and 4 female mice in the F1 progeny were mated withMCH(ICR) mice (purchased from CREA JAPAN, INC.) to obtain F2 progenies,which were subjected to PCR analysis of tail DNA and analysis for humanantibody μ chain expression as described above. As a result, it wasconfirmed that SC20 fragment was transmitted to 30% of the F2 progenythrough F1 male mice (43 out of the 142 offsprings were positive) and to33% of the F2 progeny through F1 female mice (20 out of the 60offsprings were positive).

These results show that a mouse strain was established which retains thehuman chromosome #14 fragment (containing a human antibody heavy chaingene), which expresses human antibody heavy-chains and which cantransmit the human chromosome to the subsequent generation.

(5) Stable Retention of a Human Chromosome #14 Fragment in Mice

Three F1 mice (16-5, 17-8 and 17-23 shown in Table 26) which wereobtained in Example 68, Section 4 and which retained SC20 fragment wereused in analysis for the ratio of retention of SC20 fragment in mice.The mice were injected intraperitoneally with 0.3 ml of CORCEMID (100μg/ml) and then killed by dislocation of the cervical vertebrae in aneuthanasic manner, followed by removal of the brain, liver, spleen,testis and bone marrow. All of these tissues except the bone marrow werewashed with PBS(−), cut into pieces with scissors for anatomy, givenhypotonic treatment with KCl (0.075 M) for 15 minutes, and fixed inCarnoy's fixative. Specimens were prepared using the supernatant of theCarnoy fixation by conventional methods. FISH analysis was performedusing a human chromosome-specific probe (Human COT-1 DNA) according tothe method described in a reference (Tomizuka et al., Nature Genetics,16, 133-143). As to the brain, spleen, liver and bone marrow, 30 or morenuclei in interphase were selected randomly for each of these tissues.Then, the number of nuclei in which a signal was detected (mark “+” inFIG. 39) and the number of nuclei in which a signal was not detected(mark “−” in FIG. 39) were counted to calculate the retention ratio. Thetestis were classified into the 1st meiosis phase spreads, the 2ndmeiosis phase spreads and sperms. Ten or more spreads or sperms wereselected for each group and then counting was performed in the samemanner as described above to calculate the retention ratio. As a result,all of the 3 mice exhibited a retention ratio of almost 100% in thebrain and liver. A decrease in the retention ratio was observed in thebone marrow and spleen. In the testis, a retention ratio of 80-100% wasobtained for the 1st meiosis phase spreads, and a retention ratio of30-50% for sperms. Assuming that SC20 fragment is retained stably, thetheoretical retention ratio should be 100% for the 1st meiosis phasespreads and 50% for the 2nd meiosis phase spreads and sperms. Thus, itis believed that SC20 fragment is retained stably in the testis.

At the same time, fibroblasts were prepared from the tail and then theratio of retention of SC20 fragment was examined in the same manner asin Example 79. As a result, the retention ratios in mice 16-5, 17-8 and17-23 were 98%, 96% and 98%, respectively (50 nuclear plate were testedfor each mouse).

(6) Hereditary Relief of Antibody Production Ability-Deficient Mice bythe Transfer of a Human Chromosome #14 Fragment (Containing AntibodyHeavy-Chain Gene)

The knockout mouse whose antibody μ chain gene essential for thegeneration of B lymphocytes is disrupted (Section 1, Example 67) cannotproduce antibody because the mouse is deficient in mature B lymphocytesresponsible for humoral immunity. The following experiment was conductedto examine as to whether this deficiency could be relieved bytransferring SC20 fragment (containing a human antibody heavy-chaingene) by mating.

Those mice exhibiting agouti coat color among the progeny obtained bymating the endogenous antibody heavy-chain one allele-disrupted TT2Fcell clone-derived chimeric mice from Example 49 with MCH(ICR) mice weresubjected to Southern blot analysis to select mice retaining thedisrupted allele. A female antibody heavy-chain-deficient heterozygotethus selected was mated with a male F1 offspring (17-7) which wasobtained in Example 68, Section 4 and which retains SC20 fragment. Theresultant 5 offspring mice were subjected to both PCR analysis forconfirming the retention of SC20 fragment and determination of humanantibody μ chain and γ chain in the sera (see Example 68, Section 4). Asa result, it was confirmed that three mice #2, #3 and #5 retained SC20fragment (Table 27). Furthermore, as a result of the analysis for mouseantibody A chain expression (Example 75), it was demonstrated that mice#2 and #3 are mouse μ chain-negative, that is, endogenous antibodyheavy-chain-deficient homozygotes (Table 27). These results wereconsistent with the results of Southern blot analysis (see Example 49)using the DNAs prepared from the tails of the 5 mice. Compared to mouse#1 in which neither mouse nor human antibody heavy chain was detected,very high concentrations of human antibody μ chain (310 mg/l) and γchain (860 mg/l) were detected in mouse #3 which is antibodyheavy-chain-deficient homozygote and which retains the human chromosome#14 fragment. Further, quantitative determination of human γ subclasseswas performed on mouse #3 in the same manner as in Example 29 to detectall of the 4 subclasses (γ1, γ2, γ3 and γ4). In particular, theconcentration of human μ chain in this mouse is comparable to theconcentration of mouse μ chain in wild-type mice (Mendez et al., NatureGenet. 15, 146-156 (1977)). These results show that the symptom ofinability for antibody production because of disruption of endogenousheavy-chain gene (deficiency of B lymphocytes: see Kitamura et al.,Nature, 350, 423-, 1991) in this mouse was cured by the transfer ofhuman chromosome #14 fragment SC20 (containing an antibody heavy-chaingene), and that the mouse has recovered the ability to produce antibodyand the ability to produce B lymphocytes.

TABLE 27 Mouse Retention of Mouse Human IgM Human IgG No. SC20 Fragmentμ Chain (mg/l) (mg/l) 1 − − Below detection 0.33 limit 2 + + 8.4 5.3 3 +− 310 860 4 − + Not measured Not measured 5 + + 4.8 0.86

EXAMPLE 69 Retention of the Human Chromosome in Offsprings of HumanChromosome #22 (Fragment)-Transferred ES Cell-Derived Chimeric Mice (1)Fragmentation of Human Chromosome #22 Using Microcell Fusion

Since it was observed that both a human chromosome #2 fragment (Example42) and a human chromosome #14 fragment (Example 68, Section 4) oncetransferred into mice were transmitted to their offsprings, it isexpected that fragmentation of human chromosome #22 would increase thepossibility of transmission of this chromosome to offspring mice. When ahuman chromosome is transferred into a recipient cell by microcellfusion, it is observed that 40-80% of the transferred clones retain thehuman chromosome which has been fragmented at the time of fusion(Oshimura et al., Protein, Nucleic Acid, Enzyme, vol. 35, No. 14, 1990).The present inventors tried to fragment human chromosome #22 utilizingthis phenomenon.

A microcell fusion experiment (see Example 1) was conducted using clone6-1 from Example 35 as a chromosome donor cell and wild-type mouse A9cells as a recipient cell, thereby producing seventy-three G418resistant clones. Genomic DNAs were prepared from the resultant clonesand then screened by PCR using Igλ primers (Example 2). Sixty-sevenclones which retained human Igλ gene were subjected to PCR analysisusing primers specific to 8 markers located on human chromosome #22(D22S315, D22S275, D22S280, D22S278, D22S283, D22S272, D22S282 andD22S274; for the order of location on human chromosome #22, see Nature,vol. 377, 367-379 (1995); base sequences for these primers were obtainedfrom databases of such as GenBank). As a result, it was found that apart of the markers disappeared in 25 clones. Thus, it was suggestedthat chromosome #22 was fragmented in these clones (FIG. 40). Amongthem, clone #22 and clone #28 are considered to have a fairly smallfragment, because markers other than Igλ and D22S315 disappeared in theformer and markers other than Igλ disappeared in the latter (FIG. 40).Clone #28 was subjected to FISH analysis (see Example 18) using a humanchromosome-specific probe. The results are shown in FIG. 41. It isobserved that the size of the chromosome hybridizing to the probe issmaller in this clone than in the control clone (containing an intactchromosome #22). Thus, a human chromosome #22 fragment containingantibody λ gene could be obtained as a result of fragmentation whichoccurred at the time of microcell fusion.

(2) Transfer of Chromosome #22 (Fragment) into TT2F Cells

Chromosome #22 (fragment) was microcell-transferred into TT2F cells bythe method described in Example 2, using clones #22, #28 and 6-1 aschromosome donor cells. Clones #22 and A9/#22 (6-1) were subjected topuromycin (0.75 μg/ml) selection, and clone #28 was subjected to G418(225 μg/ml) selection. As a result, drug resistant clones were obtainedas follows: 13 from clone #22, 5 from clone 6-1 and 3 from clone #28.These ES cell clones are subjected to PCR analysis and FISH analysis toconfirm the retention of human chromosome #22 (fragment) in the samemanner as in Example 69, Section 1.

(3) Production of Chimeric Mice from ES Cell Clones Retaining HumanChromosome #22 (Fragment)

In the same manner as in Example 3, chimeric mice are produced from thedrug resistant ES cell clones which were obtained in Example 69, Section2 and which were confirmed to retain human chromosome #22. Confirmationof the retention of human chromosome #22 (fragment) in the resultantchimeric mice is performed by the method described in Example 4.

(4) Transmission of Human Chromosome #22 (Fragment) to Offsprings

The chimeric mice retaining human chromosome #22 (fragment) are mixedand mated with ICR mice.

Retention of a human chromosome #22 fragment in the offsprings isexamined by PCR using genomic DNAs prepared from the tails of theoffspring mice having agouti coat color (see Examples 30, 42 and 43). Asshown in Examples 42 and 43, mouse ES cell clones retaining humanchromosome #22 or a fragment thereof can differentiate into oocytes orsperms functional in chimeric mice, thereby allowing to the humanchromosome #22 (fragment) to be transmitted their progenies. Thus, it ispossible to establish a mouse strain which retains human chromosome #22(fragment) containing human antibody light-chain λ gene and which cantransmit it to the subsequent generation.

EXAMPLE 70 Production of Mice Retaining Both Human Chromosome #2(Fragment) and #14 (Fragment) by Mating

The human chromosome #2 (fragment)-retaining mouse strain from Example42 or 43 is mated with the human chromosome #14 (fragment)-retainingmouse strain from Example 68 to produce offsprings. Genomic DNAs areprepared from the tails of the offspring mice. The DNA is analyzed byPCR, etc. (Examples 9, 42 and 43) to produce those mice which retainboth human chromosome #2 partial fragment and human chromosome #14(fragment)

EXAMPLE 71 Production of Mice Retaining Both Human Chromosome #22(Fragment) and #14 (Fragment) by Mating

The human chromosome #22 (fragment)-retaining mouse strain from Example69 is mated with the human chromosome #14 (fragment)-retaining mousestrain from Example 68 to produce offsprings. Genomic DNAs are preparedfrom the tails of the offspring mice. The DNA is analyzed by PCR, etc.(Examples 30, 42 and 43) to produce those mice which retain both humanchromosome #22 (fragment) and #14 (fragment).

EXAMPLE 72 Production of Mice Retaining the Three Human Chromosomes #2(Fragment), #14 (Fragment) and #22 (Fragment) by Mating

The mouse strain retaining both human chromosome #2 (fragment) and #14(fragment) obtained in Example 71 is mated with the mouse strainretaining a human chromosome #2 fragment obtained in Example 42 or 43 toproduce offsprings. Genomic DNAS are prepared from the tails of theoffspring mice. The DNA is analyzed by PCR, etc. (Examples 9, 30, 42 and43) to produce those mice which retain all of the three humanchromosomes, #22 (fragment), #14 (fragment) and #2 (fragment).Alternatively, mice retaining all of the above three human chromosomesmay also be obtained by mating the mouse strain retaining both humanchromosome #2 (fragment) and #14 (fragment) from Example 70, with themouse strain retaining a human chromosome #22 fragment from Example 69.

EXAMPLE 73 Production of a Complete Human Antibody-Producing MouseStrain by Mating

The mouse strains retaining human chromosomes #2+#14 (Example 70),#14+#22 (Example 71) and #2+#14+#22 (Example 72), respectively, arerepeatedly mated with a mouse strain deficient in endogenous antibodyheavy-chain and light-chain κ genes. From the resultant offsprings,those mouse strains which retain human chromosomes #2+#14, #14+#22 or#2+#14+#22 and which are homozygotes in the deficiency of endogenousantibody heavy-chain and light-chain κ genes, are selected by PCRanalysis, etc. (Examples 9, 30, 42 and 43). In these strains, completehuman antibodies are mainly produced (Green et al., Nature Genetics,7:13-, 1994; Lonberg et al., Nature, 368:856-, 1994).

Hereinbelow, the establishment of a mouse strain which retains both ahuman chromosome #2 fragment and a human chromosome #14 fragment andwhich is homozygote in the deficiency of endogenous antibody heavy-chainand light-chain κ genes will be described. The 4 strains used for themating and the method for assaying the genotypes of each strain are asfollows.

(1) The mouse strain from Example 42 retaining a human chromosome #2fragment: the retention of the human chromosome #2 fragment is assayedby PCR analysis of the tail-derived DNA as described in Example 42 andby the expression of human antibody κ chain in the sera.(2) The mouse strain from Example 68, Section 4 retaining a humanchromosome #14 fragment: the retention of the human chromosome #14fragment is assayed by PCR analysis of the tail-derived DNA as describedin Example 68, Section 4 and by the expression of human antibody g chainin the sera.(3) The antibody heavy-chain knockout mouse strain from Example 67,Section 1: heavy-chain deficiency-homozygotes or heterozygotes wereassayed by Southern blot analysis of the tail-derived DNA as describedin Example 67, Section 1 and by the presence or absence of theexpression of mouse antibody μ chain in the sera (see Example 75).(4) The antibody κ chain knockout mouse strain from Example 80: κ chaindeficiency-homozygotes or heterozygotes were assayed by Southern blotanalysis of the tail-derived DNA as described in Example 80.

A mouse strain which retains all of the 4 genotypes (i.e., retaining ahuman chromosome #2 fragment, retaining a human chromosome #14 fragment,antibody heavy-chain-deficiency homozygote or heterozygote, and antibodyκ chain-deficiency homozygote or heterozygote) was established by matingthe above 4 strains with each other. Specifically, after the above 4strains used as starting materials were mated several times, a malemouse having the genotypes of “retaining the human chromosome #14fragment, antibody heavy-chain-deficiency homozygote and antibody κchain-deficiency heterozygote” was mated with a female mouse having thegenotypes of “retaining the human chromosome #2 fragment, antibodyheavy-chain-deficiency homozygote and antibody κ chain-deficiencyhomozygote” or “retaining the human chromosome #2 fragment, antibodyheavy-chain-deficiency homozygote and antibody κ chain-deficiencyheterozygote” or “retaining the human chromosome #2 fragment, antibodyheavy-chain-deficiency heterozygote and antibody κ chain-deficiencyhomozygote”. As a result, mouse HK23 “retaining the human chromosome #2fragment, retaining the human chromosome #14 fragment, antibodyheavy-chain-deficiency homozygote and antibody κ chain-deficiencyheterozygote” and mouse HK29 “retaining the human chromosome #2fragment, retaining the human chromosome #14 fragment, antibodyheavy-chain-deficiency heterozygote or wild-type, and antibody κchain-deficiency heterozygote” were obtained. FIG. 42 shows theconcentration of each antibody in the sera and the genotypes of thesemice, together with the data on mouse HK28 which was also produced bythe above-described mating and which has the genotypes of “retaining thehuman chromosome #2 fragment, retaining the human chromosome #14fragment, antibody heavy-chain-deficiency heterozygote or wild-type, andantibody κ chain-deficiency wild-type”. A complete human antibodyconsisting of human μ chain and human μ chain was detected at aconcentration of 18 mg/l in the serum of mouse HK23 (Example 38).

It is possible to produce those mice having the genotypes of “retainingthe human chromosome #2 fragment, retaining the human chromosome #14fragment, antibody heavy-chain-deficiency homozygote and antibody κchain-deficiency homozygote” by mating the mice obtained by the abovemating with each other. In this mouse strain, it is expected that humanantibody κ chain will be expressed at a higher concentration than inmouse HK23 because the deficiency of the endogenous κ chain gene issubstituted by the human antibody κ chain gene contained in the humanchromosome #2 fragment (Lonberg et al., Nature, 368, 856-, 1994). It isalso expected that the concentration of a complete human antibodyconsisting of human heavy-chain and human κ chain will increase further.

EXAMPLE 74 Production of a Human Antibody-Producing Hybridoma from aMouse Strain which is Obtained by Mating and which Retains a HumanChromosome(s) Containing a Human Antibody Gene(s)

The mice retaining a human chromosome(s) containing a human antibodygene(s) which were obtained in Example 42, 43, 68, 69, 70, 71, 72 or 73are immunized with an antigen of interest in the same manner as inExample 25. The spleen is removed from each mice and the spleen cellsare fused with myeloma cells to produce hybridomas. After cultivationfor 1-3 weeks, the culture supernatant is analyzed by ELISA. The ELISAis performed by the method described in Examples 14, 15, 21, 22, 25, 33,34, 37 and 38. As a result, human antibody positive clones and cloneswhich are human antibody positive and specific to the antigen used inthe immunization are obtained.

EXAMPLE 75 Detection and Determination of Mouse IgM in Sera of ChimericMice Derived from the Mouse Antibody Heavy-Chain Both Alleles-DisruptedTT2F Cell Clone

Offspring mice were born in the same manner as in Example 40 from themouse antibody heavy-chain both alleles-disrupted TT2F cell clone(#131-3) from Example 51. Three mice having chimerisms of 0%, 50% and99%, respectively, were selected from the offspring mice. Mouse IgM intheir sera was detected and determined. Briefly, the chimeric mice ofabout 2 weeks after birth were bled and mouse IgM concentration in thesera was determined by ELIZA by the same procedures as in Example 14. APBS-diluted anti-mouse IgM antibody (Kirkegaard & Perry LaboratoriesInc., 01-18-03) was fixed, and then a PBS-diluted serum samplesupplemented with 5% FBS was added. Peroxidase-labeled anti-mouse IgMantibody (Kirkegaard & Perry Laboratories Inc., 074-1803) was added andthe absorbance at 450 nm was determined using TMBZ as a substrate.Purified mouse IgM (Pharmingen, 03081D) was used as a standard. Thisstandard was diluted stepwise with FBS-supplemented PBS. The results areshown in Table 28. Of the chimeric mice derived from the mouse antibodyheavy-chain both alleles-disrupted TT2F cells, the mouse having achimerism of 99% exhibited a low mouse IgM concentration. Thus, it wasconfirmed that the mouse heavy-chain gene from the ES cells hardlyfunctions in this mouse.

TABLE 28 Concentration of Mouse IgM in Chimeric Mice (ELISA) Chimerism %IgM (mg/l) 0 12 50 11 99 1.5

EXAMPLE 76 Preparation of a Targeting Vector for Knocking Out AntibodyLight-Chain κ Gene in ES Cells

Plasmid LoxP-PGKPuro in which LoxP sequence was inserted at both ends ofa puromycin resistance gene was prepared in the same manner as inExample 48, Section 1. Briefly, a puromycin resistance cassette PGKPurowas cut out from PGKPuro plasmid DNA (Watanabe et al., Biochem. Biophys.Res. Commun. 213:130-137 (1995); released from Peter W. Laird, WhiteheadInstitute for Biochemical Research and Massachusetts Institute ofTechnology, Dept. of Biology, Cambridge, Mass.) using restriction enzymeSalI and then blunted. PGKPuro was inserted into the SmaI and EcoRVrestriction sites of a LoxP-sequence containing plasmid to produceplasmid pLoxP-PGKPuro (FIG. 30). Further, a DNA fragment comprising agenomic DNA constant region containing the mouse antibody light-chain κJregion and constant region was replaced with the LoxP-PGKPuro gene inthe same manner as in Example 48 (FIG. 31).

EXAMPLE 77 Production of an Antibody Light-Chain Gene BothAlleles-Disrupted Strain from AntibodyLight-Chain-Deficient-Heterozygote (and AntibodyHeavy-Chain-Deficient-Homozygote) Mouse ES Cells

(1) a Puromycin Resistance Gene was Inserted into the AntibodyLight-Chain Deficient-Heterozygote TT2F Clone (HD43) Obtained in Example58 to Give a Strain in which both Alleles of a Light-Chain Gene wereDisrupted.

The antibody light-chain targeting vector prepared in Example 76 waslinearized with restriction enzyme KpnI to transform HD43 clone in thesame manner as in Example 58. The resultant transformants were subjectedto selective culture at a puromycin concentration of 0.75 g/ml. At day7-9 of the cultivation, colonies formed were picked up. A part of thesecolonies was stored frozen, and the remaining part was used to preparegenomic DNA in the same manner as in Example 49. Genomic DNAs from thepuromycin resistant strains were digested with restriction enzyme EcoRI(Takara Shuzo), separated by agarose gel electrophoresis and subjectedto Southern blot analysis to detect homologous recombinants using theprobe described in Example 48, Section 4 (see Examples 58 and 59). As aresult, 4 clones in which both alleles of an antibody light-chain weredisrupted were obtained from the 74 clones analyzed. Under usual cultureconditions, no changes in growth rate and morphology were observed inthese clones, as compared to the TT2F clone before gene disruption. Thissuggests that the clones under consideration retain the ability toproduce chimera.

(2) Production of Chimeric Mice from the AntibodyHeavy-Chain-Deficient-Homozygote and Antibody Light-Chain Gene BothAlleles-Disrupted Clone

Cells in the frozen stock of antibody light-chain gene bothalleles-disrupted TT2F cell clone HD43P-10 from Example 77, Section 1were thawed, started up for culture and injected into 8-cell stageembryos obtained by mating male and female mice of ICR (CREA JAPAN,INC.); the injection rate was 10-12 cells per embryo. After the embryoswere cultured overnight in the medium for ES cells (see Example 9) todevelop into blastocysts, about 10 of the injected embryos weretransplanted to each side of the uterus of foster mother ICR mice (CREAJAPAN, INC.; 2.5 days after pseudopregnant treatment).

As a result of transplantation of a total of 161 injected embryos, 37offspring mice were born. Chimerism in the offsprings can be determinedby the extent of TT2 cell-derived agouti coat color (dark brown) in thehost embryo (ICR)-derived albino coat color (white). Out of the 37offsprings, 9 mice were recognized to have partial agouti coat color,indicating the contribution of the ES cells. Out of the 9 mice, fourwere chimeric mice in which more than 80% of their coat color was agouti(i.e. ES cell-derived).

From these results, it was confirmed that antibody light-chain bothalleles-disrupted ES cell clone HD43P-10 maintains a high ability toproduce chimera.

EXAMPLE 78 Removal of the G418 Resistance and Puromycin ResistanceMarker Genes from the Antibody Light-Chain Deficient-Homozygote (andAntibody Heavy-Chain Deficient-Homozygote) TT2F Cell Clone

From the antibody light-chain both alleles-disrupted HD43P-10 clone(puromycin resistant, G418 resistant) which was obtained and confirmedto have a high chimera-forming ability in Example 77, the puromycinresistance and G418 resistance marker genes were removed by theprocedures described in Example 52. Briefly, an expression vector pBS185(BRL) containing a Cre recombinase gene which causes a site-directedrecombination between the LoxP sequences inserted at both ends of theG418 resistance marker gene was transferred into the clone describedabove in the same manner as in Example 52. The resultant puromycin (0.75μg/ml) sensitive clones (6 clones) were grown to confluence in 35 mmplates in the same manner as in Example 52. Three fifths (⅗) of theresultant culture were suspended in 0.5 ml of a preservation medium [ESmedium+10% DMSO (Sigma)] and stored frozen at −80° C. The remaining twofifth (⅖) were divided into two portions and inoculated into two 12-wellgelatin-coated plates. Cells in one plate were cultured in non-selectivemedium for 2 days. Cells in other plate were cultured in the presence of300 μg/ml of G418 for 2 days. As a result, 5 puromycin sensitive andG418 sensitive clones which would be killed in the presence of G418 wereobtained.

EXAMPLE 79 Increase in the Expression of Human Antibody κ Chain in Seraas a Result of Mating a Human Chromosome #2 Fragment-Retaining MouseStrain with C57BL/6 Strain

The hereditary background of the progenies of the chimeric mice[hereinafter referred to as “F1(chimera×MCH)”] which were described inExamples 43 and 44 and which retain a human chromosome #2 fragment(hereinafter referred to as “W23 fragment”) is that they are a mixtureof TT2F cell (Example 39)-derived CBA mouse strain and C57BL/6 mousestrain, and MCH(ICR) mouse strain mated with the chimeric mice. In orderto observe the behavior of W23 fragment under a hereditary background ashomogeneous as possible, first, F1(chimera×MCH) were back-crossed withMCH(ICR). The offspring mice obtained by the mating of F1(chimera×MCH)(randomly selected 8 male and 6 female mice)×MCH(ICR) were examined asto whether they would retain W23 fragment in the same manner as inExample 43. As a result, it was confirmed that W23 fragment wastransmitted through male to 8% of the offsprings (25 out of the 324offsprings were positive) and through female to 22% of the offsprings(32 out of the 148 offsprings were positive). When the resultantF2(F1×MCH) (randomly selected 8 male and 8 female mice) were furthermated with MCH(ICR), the transmission ratio was 9% through male (30 outof the 346 offsprings were positive) and 24% through female (48 out ofthe 202 offsprings were positive). Thus, the results was similar to thatobtained by the mating of F1(chimera×MCH)×MCH(ICR). F3(F2×MCH) wereobtained by the latter mating.

The concentrations of human antibody κ chain in the sera of 4-12-weekold chimeric mice (FIG. 43, indicated as “Chimera”, 4 mice),F1(chimera×MCH) (19 mice), F2(F1×MCH) (39 mice) and F3(F2×MCH) (33 mice)were determined in the same manner as in Example 44. The results areshown in FIG. 43. Human antibody κ chain was detected in all of the miceretaining W23 fragment. On the other hand, the κ chain concentrationsvaried greatly in F2(F1×MCH) and F3(F2×MCH); the averaged values inthese groups were lower than those in the chimeric mice andF1(chimera×MCH).

In order to examine the influence which would be caused by the matingwith a strain other than MCH(ICR), the same F2(F1×MCH) mice as used inthe experiment of mating with MCH(ICR) were mated with C57BL/6N(purchased from CREA JAPAN, INC.). Concentrations of κ chain weredetermined in the same manner on the resultant 26 mice retaining W23fragment [F3(F2×C57BL/6)]. As a result, these mice exhibited κ chainconcentrations as high as those in the chimeric mice and F1(chimera×MCH)(FIG. 43). As described above, F3 (F2×MCH) and F3 (F2×C57BL/6) arederived from the same F2(F1×MCH) mice as one of the parents. Therefore,it is believed that the difference between F3 (F2×MCH) andF3(F2×C57BL/6) is in their hereditary background alone. Thus, it isindicated that difference in hereditary background influences the amountof expression of human antibody κ chain. Further, it has become clearthat the hereditary background of C57BL/6 is more desirable than that ofMCH(ICR) for efficient expression of human antibody κ chain. Fromsimilar experiments, it has been demonstrated that the hereditarybackground of C3H HeN (purchased from CREA JAPAN, INC.) is comparable toor better than that of C57BL/6 for efficient expression of humanantibody κ chain.

The following experiment was conducted to examine as to whether theinfluence of hereditary background on antibody κ chain concentrationsobserved above is related to the ratio of chromosome retention(stability) at the level of individual mice. Briefly, metaphasechromosome samples were prepared from tail-derived fibroblasts and bonemarrow cells of 2F-1 mouse (serum κ chain concentration: 84 mg/l) and1F-3 mouse (serum κ chain concentration: 13 mg/l) in F1(chimera×MCH) andsubjected to FISH analysis (Tomizuka et al., Nature Genetics, vol 16,133-143). The ratio of those metaphase spreads containing W23 fragmenthybridizing to a human chromosome-specific probe to all of the spreadsobserved was determined. It is believed that the resultant valuesrepresent the W23 fragment retention ratios in fibroblasts and bonemarrow cells, respectively. As a result, with respect to 2F-1, theretention ratio was 51% in fibroblasts and 34% in bone marrow cells; andwith respect to IF-3, the retention ratio was 23% in fibroblasts and 18%in bone marrow cells (more than 50 nuclear plate were measured for eachcase). These results suggest that the κ chain concentrations in seracorrelated to the ratios of retentions of W23 fragment in fibroblastsand bone marrow cells. In other words, it is very likely that hereditarybackground influences the stability of the transferred human chromosomefragment itself. Thus, it is believed that the hereditary background ofC57BL/6 or C3H strain is desirable for efficient expression of a genenot only on the chromosome #2 fragment described herein but also onother chromosome fragments (e.g. chromosome #14 fragment).

In order to verify the above conjecture, a male mouse #17-7 inF1(chimera×MCH) which retains the human chromosome #14 fragment obtainedin Example 68 (hereinafter referred to as “SC20 fragment”) and whichexpresses a human antibody heavy-chain in the serum was mated withMCH(ICR) and C57BL/6. Of the resultant offsprings, two F2(F1×MCH) miceand two F2(F1×C57BL/6) mice, each retaining SC20 fragment, weresubjected to determination of human antibody heavy-chain concentrationsin the sera (see Example 68). Furthermore, metaphase chromosome sampleswere prepared from the tails of these mice and then the ratio of SC20fragment retention was determined in the same manner as for W23fragment. As a result, the human μ chain concentration was 11.0 mg/l and1.1 mg/l and the chromosome retention ratio 74% and 54% in F2(F1×MCH)whereas the human μ chain concentration was 47 mg/l and 54 mg/l and thechromosome retention ratio 84% and 88% in F2 (F1×C57BL/6).F2(F1×C57BL/6) mice exhibited higher values in both the human μ chainconcentration and the chromosome retention ratio. Thus, it has becomeclear that the hereditary background of C57BL/6 is desirable for stableretention of a transferred human chromosome and for efficient expressionof a gene located thereon, as presumed from the results obtained on W23fragment-retaining mice.

EXAMPLE 80 Production of Chimeric Mice from an AntibodyHeavy-Chain-Deficient and Antibody κ Chain-Homologous Recombinant ESCell Clone

Cells in the frozen stock of antibody heavy-chain-deficient and antibodyκ chain-homologous recombinant ES cell clone HD43 from Example 58 werethawed, started up for culture and injected into 8-cell stage embryosobtained by mating male and female mice of ICR (CREA JAPAN, INC.); theinjection rate was 10-12 cells per embryo. After the embryos werecultured overnight in the medium for ES cells (see Example 9) to developinto blastocysts, about 10 of the injected embryos were transplanted toeach side of the uterus of foster mother ICR mice (CREA JAPAN, INC.; 2.5days after pseudopregnant treatment).

As a result of transplantation of a total of 314 injected embryos, 51offspring mice were born. Chimerism in the offsprings can be determinedby the extent of TT2 cell-derived agouti coat color (dark brown) in thehost embryo (ICR)-derived albino coat color (white). Out of the 51offsprings, 26 mice were recognized to have partial agouti coat color,indicating the contribution of the ES cells. Out of the 26 mice, twowere chimeric female mice in which 100% of their coat color was agouti(i.e. ES cell-derived)

From these results, it was confirmed that antibody heavy-chain-deficientand antibody light-chain-homologous recombinant ES cell clone HD43maintains the ability to produce chimera. In the female mice exhibiting100% contribution, it is highly possible that the ES cells have beendifferentiated into functional germ cells (oocytes).

Examination was made as to whether ES cell-derived offsprings would beproduced by mating the above female chimeric mice (both having 100%chimerism in coat color) with male ICR mice. By this mating, offspringswith agouti coat color should be produced from TT2F cell (agouti:dominant)-derived oocytes in the chimeric mice fertilized by male ICRmouse (albino: recessive)-derived sperms, and offsprings with albinocoat color should be produced from ICR-derived oocytes. Actually, all ofthe viable offspring mice obtained by one mating for each female mouseexhibited ES cell-derived agouti coat color. Genomic DNAs were preparedfrom the tails of these offspring mice to examine the presence of anantibody κ chain disrupted allele by Southern blot analysis (Example58). As a result, mice having an antibody κ chain disrupted allele wereobtained.

Twenty-seven offspring mice produced by the mating of the antibodylight-chain deficient-heterozygote male and female mice were subjectedto Southern blot analysis (Example 58). As a result, antibodylight-chain wild-type alleles disappeared and only disrupted alleleswere observed in 7 offspring mice. Hence, these mice were believed to beantibody light-chain-deficient homozygotes. FIG. 44 shows the results ofdetection and quantitative determination of mouse antibody κ chain and λchain in the sera.

In those mice which were judged to be antibody light-chain-deficienthomozygotes by the Southern blot analysis (Nos. 4, 6, 14, 22, 24, 25 and26 in FIG. 44), the concentrations of κ chain are greatly reduced (theremaining κ chain appears to be derived from their mother mice).Instead, the concentrations of λ chain are greatly increased in thesemice. These results are consistent with the reported results of analysisof the antibody κ chain knockout mouse (Yong-Rui Zou et al., EMBO J. 12,811-820 (1993)).

Thus, an antibody κ chain knockout mouse strain could be establishedfrom antibody κ chain homologous recombinant ES cell clone HD43.

EXAMPLE 81 Preparation of a Targeting Vector for Inserting HumanTelomere Sequence into Human Chromosome #22

Fragmentation of human chromosome #22 on which human antibody λ chaingene (hereinafter referred to as “Igλ gene”) was located was attemptedby inserting human telomere sequence by homologous recombination (J. E.Itzhaki et al., Nature Genet., 2, 283-287, 1992). Specifically, atargeting vector for inserting human telomere sequence into the LIFlocus located very close to Igλ gene (on the telomere side) wasprepared.

Human telomere sequence was synthesized by PCR according to the methodof J. J. Harrington et al. (Nature Genet., 15, 345-355, 1997). The PCRproduct was purified by agarose gel electrophoresis and then bluntedwith DNA Blunting Kit (Takara Shuzo). The blunted PCR product wasinserted into the Eco RV site of pBluescript SK II(+) (Toyobo) byligation using DNA Ligation Kit (Takara Shuzo) (pBS-TEL). This plasmidpBS-TEL was sequenced. As a result, it was found that the telomeresequence had been inserted in the following direction: HindIII-(TTAGGG)N-Eco RI.

Subsequently, the LIF gene region on human chromosome #22 to be used inthe homologous recombination was amplified by PCR as described below,and then cloned into plasmid PBS-TEL. The sequences of the primers usedin the PCR were as follows.

Sense primer: (SEQ ID NO:65) 5′-TCGAACTAGTAGGAGAAGTGAACTTGAGGAGGC-3′Antisense primer: (SEQ ID NO:66) 5′-TCGAACTAGTGATTCAGTGATGCTGTGCAGG-3′

The PCR reaction mixture was composed of 5 μl of 10×LA PCR buffer II(Mg²⁺ free) (Takara Shuzo); 5 μl of 25 mM MgCl2; 8 μl of dNTP mixture(2.5 mM each) (Takara Shuzo); 10 μmol of sense primer; 10 μmol ofantisense primer; 100 ng of template DNA (HFL1, genomic DNA from primaryculture human fibroblasts); 0.5 μl of LA Taq (5 U/μl) (Takara Shuzo) andsterile distilled water to make a total volume of 50 μl. All of theoperations for preparing the reaction mixture were carried out on ice.Then, reaction tubes were placed in the well of a thermal cycler (PCRSystem 9600, Perkin-Elmer) preset at 85° C. After the tubes were heatedat 94° C. for 1 minute, 35 cycles of reaction were carried out at 98° C.for 10 seconds and at 65° C. for 5 minutes. The PCR product was purifiedand then digested with Spe I (Spe I site was present in the primers),followed by insertion into the Spe I site in PBS-TEL. The plasmid inwhich the LIF gene had been inserted in a opposite direction of thehuman telomere sequence (TTAGGG)n was selected (M. Giovannini et al.,Cytogenet Cell Genet. 64, 240-244, 1993) (pBS-TEL/LIF).

Subsequently, plasmid pGKpuro containing a puromycin resistance gene (S.Watanabe et al., Biochem. Biophys. Res. Comm., 213, 130-137, 1995) wasdigested with Eco RI and blunted, followed by insertion of Not I linker.The puromycin resistance gene was cut out by digesting the resultantplasmid with Not I and then inserted into the Not I site of pBS-TEL/LIF.The plasmid in which the direction of transcription of the puromycinresistance gene was the same as that of the LIF gene was selected(pBS-TEL/LIFPuro, see FIG. 45). The resultant plasmid was amplified inE. coli DH5, purified with QUIAGEN column (Funakoshi) and used fortransfection (as described later).

EXAMPLE 82 Transfer of Human Chromosome #22 into Chicken DT40 Cells

Mouse A9 cells containing human chromosome #22 marked with a G418resistance gene (Tomizuka et al., Nature Genet., vol 16, 133-143, 1997;hereinafter referred to as “A9/#22neo”) were cultured in Dulbecco'smodified Eagle's Minimal Essential Medium (hereinafter referred to as“DMEM”) supplemented with 10% fetal bovine serum and G418 (800 μg/ml).Chicken DT40 cells were cultured in DMEM supplemented with 10% FBS, 1%chicken serum and 10⁻⁴ M 2-mercaptoethanol.

Microcells were prepared as described below (for details, see Shimizu etal., “Cell Technology Handbook”, published by Yodosha, p. 127-).

A9/#22neo cells were cultured in twelve 25 cm² centrifuge flasks(Costar) until the cell concentration reached about 90% saturation.Then, the medium was exchanged with a medium (DMEM+20% FBS) supplementedwith COLCEMID (0.07 μg/ml; demecolcine, Wako Pure Chemical Industries,Inc.).

The cells were cultured for another 2.5-3 days to form microcells.Thereafter, the culture solution was removed from the centrifuge flasks,into which a solution of cytochalasin B (10 μg/ml, Sigma) prewarmed at37° C. was filled and centrifuged at 34° C. at 8000 rpm for 1 hour. Themicrocells were suspended in DMEM and purified by filtration withfilters. After the purification, the microcells were centrifuged at 1500rpm for 10 minutes and then suspended in 5 ml of DMEM. DT40 cells(2×10⁷) were centrifuged at 1000 rpm for 5 minutes, washed with DMEMtwice and suspended in 5 ml of DMEM. The microcells prepared above werere-centrifuged at 1500 rpm for 10 minutes and then, without removal ofthe supernatant, 5 ml of the previously prepared DT40 suspension wasoverlayered gently. After centrifugation at 1300 rpm for 5 minutes, thesupernatant was removed. The cell pellet was suspended in 2 ml of PHA-P(100 μg/ml, DIFCO) and left to stand in an incubator at 37° C. under 5%CO2 for 15 minutes. Then, the suspension was centrifuged at 1700 rpm for7 minutes. The supernatant was removed and the cell pellet was loosenedby tapping. To the cell pellet, 1 ml of PEG1500 (polyethylene glycol,Boehringer) was added gently and the pellet was treated for 1.5-2minutes under agitation. After this treatment, 1 ml of DMEM was addedover approximately 1 minute. Then, 3 ml of DMEM was added overapproximately 2 minutes. Thereafter, DMEM was added to make a totalvolume of 11 ml and the resultant mixture was mixed gently. The mixturewas left to stand for 10 minutes at room temperature and thencentrifuged at 1300 rpm for 5 minutes. The supernatant was removed. Thecells were suspended in 10 ml of the above-described culture medium andcultured in 0100 mm plates for 24 hours. Twenty-four hours later, themedium was exchanged with one supplemented with G418 (1 mg/ml). Theresultant culture was dispensed into three 24-well plates (SumitomoBakelite), followed by selective culture for about 2 weeks to isolateG418 resistant clones.

(1) PCR Analysis

As a result of the selective culture, about thirty G418 resistant cloneswere obtained. Genomic DNAs were extracted from these clones usingPuregene DNA Isolation Kit (Gentra System). Using the genomic DNA as atemplate, PCR was performed with human Igλ gene-specific primers toidentify clones having human chromosome #22 containing Igλ gene. The Igλgene-specific primers used were as follows.

5′-GAGAGTTGCAGAAGGGGTGACT-3′ (SEQ ID NO:67) 5′-GGAGACCACCAAACCCTCCAAA-3′(SEQ ID NO:68)

The PCR reaction mixture was composed of 5 μl of 10×Ex Taq buffer(Takara Shuzo); 8 μl of dNTP mixture (2.5 mM each) (Takara Shuzo); 10μmol of each primer; 100 ng of genomic DNA; 0.5 μl of Ex Taq (5 U/μl)(Takara Shuzo) and sterile distilled water to make a total volume of 50μl. All of the operations for preparing the reaction mixture werecarried out on ice. Then, reaction tubes were placed in the well of athermal cycler (PCR System 9600, Perkin-Elmer) preset at 85° C. Afterthe tubes were heated at 94° C. for 1 minute, 35 cycles of reaction werecarried out at 98° C. for 10 seconds, at 56° C. for 30 seconds and at72° C. for 30 seconds. As a result, 2 clones having human Igλ gene wereidentified. The presence of polymorphic markers (D22S315, D22S280,D22S283 and D22S274; Polymorphic STS Primer Pair, BIOS; J. E. Collins etal., Nature 377 suppl.: 367, 1995) located on human chromosome #22 weredetected in these clones by PCR (FIG. 46). The PCR conditions were thesame as used for the detection of human Igλ gene. Mark “◯” indicatesthat the marker was detected. Mark “X” indicates that the marker was notdetected. The diagram at the left side shows the location of each markeron chromosome #22 based on a physical map. From these results, it wassuggested that these 2 clones have a almost intact human chromosome #22.As to the other clones, although human Igλ gene was not detected, someof the polymorphic markers on chromosome #22 described above weredetected.

(2) FISH Analysis

One of the above 2 clones (clone No. 52-18) was subjected to FISHanalysis to examine how the human chromosome #22 actually existed incells. Basic operations such as preparation of chromosome samples,hybridization and detection were performed according to Tomizuka et al.(Nature Genet. 16, 133-143, 1997). As a probe, human COT-1 DNA (labeledwith Rhodamine) was used. As a result of observation of 20-30 spreads,it was confirmed that an almost intact human chromosome #22 was presentindependently (FIG. 50). Those stained in red are human chromosome #22.

From these results of analysis, it was thought that chicken DT40 cellclone 52-18 (hereinafter referred to as “DT40/#22neo”) has intact humanchromosome #22.

EXAMPLE 83 Targeted Truncation of Human Chromosome #22 in Chicken DT40Cells

DT40/#22neo from Example 82 was transfected with plasmid pBS-TEL/LIFPuroprepared in Example 81 and an attempt was made to perform targetedtruncation of the human chromosome #22 on the LIF locus.

DT40/#22neo cells were cultured under the same conditions as describedin Example 82 in the presence of G418 (1 mg/ml). 10 cells were washedwith cold PPS once, suspended in 0.5 ml of PBS and placed on ice. Then,25-30 μg of pBS-TEL/LIFPuro linearized with Eco RI was added to thecells, mixed with a pipette, transferred into an electroporation cuvette(Bio-Rad) and left to stand in ice for 10 minutes. The cuvette was setin a gene pulser (Bio-Rad) and then a voltage of 550 V was applied at acapacitance of 25 μF. After the cuvette was left to stand on ice for 10minutes, the cells were transferred into 72 cm² culture flaskscontaining the above-described medium and cultured for 24 hours.Twenty-four hours later, the medium was exchanged with a mediumsupplemented with G418 (1 mg/ml) and puromycin (0.3 μg/ml, Sigma). Theresultant culture was dispensed into five to eight 96-well cultureplates, followed by selective culture for about 2 weeks to isolateresistant clones.

(1) PCR Analysis

As a result of the selective culture, about 80 resistant clones wereobtained. Genomic DNAs were extracted from these cells as describedabove and subjected to PCR to identify homologous recombinants in whicha human telomere sequence was integrated into the LIF locus. One of theprimers was designed such that its sequence was complementary to a partof the LIF gene region which was not contained in the vector (see FIG.47). The other primer was designed such that its sequence wascomplementary to a part of the puromycin resistance gene which wascontained in the vector. The sequences of the primers are as follows.

Puro.1: 5′-GAGCTGCAAGAACTCTTCCTCACG-3′ (SEQ ID NO: 69) LIF1:5′-ATGACTCTAAGGCAGGAACATCTGTACC-3′ (SEQ ID NO: 70)

The PCR reaction mixture was composed of 5 μl of 10×LA PCR buffer II(Mg²⁺ free) (Takara Shuzo); 5 μl of 25 mM MgCl₂; 8 μl of dNTP mixture(2.5 mM each) (Takara Shuzo); 10 μmol of each primer; 100 ng of templateDNA; 0.5 μl of LA Taq (5 U/μl) (Takara Shuzo) and sterile distilledwater to make a total volume of 50 μl. All of the operations forpreparing the reaction mixture were carried out on ice. Then, reactiontubes were placed in the well of a thermal cycler (PCR System 9600,Perkin-Elmer) pre-set at 85° C. After the tubes were heated at 94° C.for 1 minute, 35 cycles of reaction were carried out at 98° C. for 10seconds and at 65° C. for 10 minutes. A 6.3 kb PCR product as shown inFIG. 47 should be detected only in the homologous recombinants ofinterest. As a result of the PCR, this 6.3 kb band was detected in 8clones (homologous recombination ratio: about 10%). When this PCRproduct was digested with Sal I, a cut pattern was obtained in exactlythe same way as expected.

Thus, it was confirmed that these 8 clones were homologous recombinants.

Subsequently, whether or not the truncation occurred as expected inthese 8 clones was examined by PCR detection of the presence of genes(Igλ, LIF, MB, IL2RB, CYP2D6, DIA1, ECGF1 and ARSA; J. E. Collins etal., Nature 377 suppl: 367, 1995) and polymorphic markers (D22S315,D22S275, D22S280, D22S281, D22S277, D22S278, D22S283, D22S272, D22S282and D22S274; J. E. Collins et al., Nature 377 suppl.: 367, 1995) onchromosome #22.

A part of the primer sequences used is as described below. The remainingprimer sequences were the same as used by Tomizuka et al. (Nature Genet.16, 133-143, 1997). The presence of LIF is evident from the experimentdescribed above.

CYP2D6 Sense primer: 5′-CTGCGTGTGTAATCGTGTCC-3′ (SEQ ID NO:71) Antisenseprimer: 5′-TCTGCTGTGAGTGAACCTGC-3′ (SEQ ID NO:72) ECGF1 Sense primer:5′-AGGAGGCACCTTGGATAAGC-3′ (SEQ ID NO:73) Antisense primer:5′-TCACTCTGACCCACGATACAGC-3′ (SEQ ID NO:74)

The PCR reaction mixture was composed of 5 μl of 10×Ex Taq buffer(Takara Shuzo); 8 μl of dNTP mixture (2.5 mM each) (Takara Shuzo); 10pmol of each primer; 100 ng of genomic DNA; 0.5 μl of Ex Taq (5 U/μl)(Takara Shuzo) and sterile distilled water to make a total volume of 50μl. All of the operations for preparing the reaction mixture werecarried out on ice. Then, reaction tubes were placed in the well of athermal cycler (PCR System 9600, Perkin-Elmer) pre-set at 85° C. Afterthe tubes were heated at 94° C. for 1 minute, 35 cycles of reaction werecarried out at 98° C. for 10 seconds, at 56° C. (65° C. for CYP2D6 andECGF1) for 30 seconds and at 72° C. for 30 seconds. The results areshown in FIG. 48. Marks “◯” and “X” have the same meanings as describedabove. As is clear from this Figure, none of the genes and markerslocated on the telomere side of the LIF locus into which a humantelomere sequence had been integrated were detected in clones 67, 68,328 and 343. It is suggested that truncation by the integration of atelomere sequence did occur as expected in at least those 4 clones.

(2) FISH Analysis

Whether the human chromosome #22 had been actually truncated or not wasexamined by FISH analysis. The experimental method was the same asdescribed above. As probes, human COT1 DNA (labeled with Rhodamine) andplasmid pGKPuro (labeled with FITC) were used. By COT1 staining, thehuman chromosome #22 can be visually checked for truncation incomparison with DT40/#22neo having intact human chromosome #22.Furthermore, if the chromosome #22 is truncated as expected on the LIFlocus into which the vector has been integrated, a signal from the Puroprobe should be detected at one end of the telomere of the chromosome#22 fragment. A part of the results is shown in FIG. 49. As a result ofobservation of 20-30 spreads for each clone, a small fragment of humanchromosome #22 (red) having a Puro probe-derived signal (yellow green)at one end of the telomere was surprisingly observed in all of the 8homologous recombinant clones. As for clones 64, 212, 222 and 305 whichwere presumed not to have undergone truncation from the results of thePCR analysis, cells having intact chromosome #22 occupied about 10% ofall cells.

These experimental results show that homologous recombinants in which ahuman telomere sequence has been integrated into the LIF locus can beobtained at an efficiency of about 10% in chicken DT40/#22neo cells andthat truncation of the human chromosome #22 has occurred at theintegration site in all of the homologous recombinants (efficiency100%).

EXAMPLE 84 Preparation of a Complete Human Antibody-Producing Hybridomafrom a Chimeric Mouse Produced by Transferring an Endogenous AntibodyHeavy-Chain and Light-Chain-Deficient Mouse ES Cells, which RetainsPartial Fragments of Human Chromosomes 14 and 22, into anImmunodeficient Mouse Host Embryo (1) Preparation of Anti-TNF-α HumanIgM Antibody

The chimeric mouse CLH13-7 (derived from TT2FES clone LH13, 50%chimerism) as produced in Example 67-(3) was immunized with human TNF-α,thereby producing hybridomas. Human Tumor Necrosis Factor-α (TNF-α,PEPRO TECH EC LTD., 300-01A) dissolved in PBS was mixed with adjuvant(MPL+TDM Emulsion, RIBI Immunochem Research Inc., R-700) to prepare0.025 mg/ml of a TNF-α solution. 0.2 ml of TNF-α solution was used forimmunization by intraperitoneal injection. Immunization has beenperformed for 70 days at an interval of 1 or 2 weeks. Two weeks afterfinal immunization, the chimeric mouse was immunized with 0.2 ml of0.025 mg/ml TNF-α dissolved in PBS. Three days after final immunization,the spleen was excised from the chimeric mouse, followed by cell fusionwith PEG according to Example 24, thereby producing hybridomas. Thefused cells were diluted to achieve 250 thousand spleen cells/ml in amedium (Sanko Junyaku Co., Ltd. S-Clone Cloning Medium CM-B)supplemented with 1 mg/ml G418, 2% fetal calf serum, and 5% HCF (AirBrown). 100 μl of the diluted cells was added to each well of a 96-wellplate and cultured. On day 14 after culturing, colonies appeared inabout 40% of the wells. The culture supernatant was analyzed by ELISAaccording to Example 14, followed by screening for a humanantibody-producing hybridoma. TNF-α was diluted to 0.5 μg/ml withcarbonic acid buffer (Sigma, C-3041), and then dispensed, 50 μl eachwell, into a 96-well plate (NUNC, 442404) for coating. Detection wasmade by TMBZ (DAKO, S1599) using biotin-labeled anti-humanimmunoglobulin λ chain antibodies (Vector, BA-3070) and an ABC kit(Vector, PK4000). The results were determined using an absorbance as anindex, approximately twice or more intense than the negative control.Cells in the positive wells were transferred into a 96-well plate, andthen cultured using a medium containing IMDM supplemented with 1 mg/mlG418 and 10% FBS. The culture supernatant was analyzed by ELISA. TNF-αwas diluted to 0.5 μg/ml with carbonic acid buffer, and then dispensed50 μl per well of a 96-well plate for coating. Detection was made byTMBZ using peroxidase-labeled anti-human immunoglobulin μ chain antibody(Biosorce, AHI0604). The results were determined using an absorbance asan index, approximately twice or more intense than the negative control.The cells in two positive wells showing strong coloration were cloned bythe limiting dilution method in the same manner as in Example 66,thereby obtaining hybridomas producing complete human antibodies thatbind to TNF-α and have human Igμ and Igλ.

(2) Preparation of Anti-TNF-α Human IgM Antibody

The chimeric mouse CLH13-13 (derived from TT2FES clone LH13, 50%chimerism) as produced in Example 67-(3) was immunized with human TNF-α,thereby producing hybridomas. Human Tumor Necrosis Factor-α (TNF-α,PEPRO TECH EC LTD., 300-01A) dissolved in PBS was mixed with adjuvant(MPL+TDM Emulsion, RIBI Immunochem Research Inc.) to prepare 0.025 mg/mlTNF-α solution. 0.2 ml of the TNF-α solution was used for immunizationby 5 times intraperitoneal injection at 1 week interval. Two weeks afterthe fifth immunization, the TNF-α solution was used to boost theimmunization. Two weeks after boosting, the chimeric mouse was immunizedwith 0.025 mg/ml TNF-α solution dissolved in PBS. The blood wascollected every week, and the anti-TNF-α human Igγ concentration inserum was detected by ELISA according to Example 14. As shown in FIG.50, an increased human Igγ antibody titer against TNF-α was shown. Threedays after final immunization, the spleen was excised from the chimericmouse, followed by cell fusion with PEG according to Example 24, therebyproducing hybridomas. The fused cells were diluted to achieve 250thousand spleen cells/ml in a medium (Sanko Junyaku Co., Ltd. S-CloneCloning Medium CM-B) supplemented with 1 mg/ml G418, 2% fetal calfserum, and 5% HCF (Air Brown). 50 μl of the diluted cells was added toeach well of a 96-well plate and cultured. On day 14 after culturing,colonies appeared in about 60% of wells. The culture supernatant wasanalyzed by ELISA according to Example 14 followed by screening for ahuman antibody-producing hybridoma. TNF-α was diluted to 0.5 μg/ml withcarbonic acid buffer, and then dispensed, 50 μl per well, into a 96-wellplate for coating. Detection was performed by TMBZ usingperoxidase-labeled anti-human immunoglobulin γ chain antibody (Sigma,A-0170). The results were determined using an absorbance as an index,approximately twice or more intense than the negative control. Cells inthe positive wells were transferred into a 96-well plate, and thencultured in a medium containing IMDM supplemented with 1 mg/ml G418 and10% FBS. The culture supernatant was analyzed by ELISA. TNF-α wasdiluted to 0.5 μg/ml with carbonic acid buffer, and then dispensed, 100μl to each well of a 96-well plate for coating. Detection was made byTMBZ using peroxidase-labeled anti-human immunoglobulin λ chain antibody(Southern Biotechnology Associates Inc., 2070-05). The results weredetermined using an absorbance as an index, approximately twice or moreintense than the negative control. The presence of complete humanantibodies containing human Igγ and Igλ was confirmed in two wells.

The result suggests that in a chimeric mouse produced by transferringendogenous antibody heavy chain- and light chain-deficient mouse EScells retaining partial fragments of human chromosomes 14 and 22 into ahost embryo of an immunodeficient mouse, increases the antibody titer ofantigen-specific human Igγ against boosting with TNF-α antigen.Furthermore, it was confirmed that the hybridoma producing a completehuman antibody IgM or IgG specific to TNF-α can be obtained from thesechimeric mice.

(3) Preparation of Human IgM Antibody Against Sugar Chain Antigen

The chimeric mice CLH13-10 (derived from TT2FES clone LH13, 60%chimerism) and CLH13-18 (derived from TT2FES clone LH13, 30% chimerism)produced in Example 67-(3) were immunized with ganglioside. An adjuvant(MPL+TDM Emulsion, RIBI Immunochem Research Inc., R-700) was dissolvedin 2 ml of the mixture of chloroform and methanol (2:1) 1 ml of eachsolution was mixed with 1 mg of GM2 (Sigma, G8397) dissolved in 1 ml ofa mixture of chloroform and methanol (2:1) or with 1 mg of ASIALO-GM1(ISOSEPAB, 65/12). The mixture was transferred into a round-bottomflask, and then dried with a rotary evaporator. 1 ml of PBS was added toeach product, and then the solution was vigorously stirred using aVortex mixer, thereby preparing a suspension. The suspension containingGM2 and that containing ASIALO-GM1 were mixed in equal quantities. Thechimeric mouse CLH13-18 was immunized intraperitonealy, and the chimericmouse CLH13-10 was immunized subcutaneously, both with 0.2 ml of themixture three times a week. The blood was collected every week, ELISAwas performed according to Example 14, so that an increase in theantibody titer was confirmed. Ganglioside GM2 or ASIALO-GM1 wasdissolved in ethanol to 6 μg/ml, and then 50 μl of each solution wasdispensed into a ELISA plate. The solution was allowed to stand for 2hours at room temperature, and then dried. A control was prepared byadding only ethanol (without ganglioside) to a plate. PBS supplementedwith 2% bovine serum albumin (BSA, Oriental yeast) was added 200μl/well, thereby blocking for 3 hours at room temperature. The sera ofthe chimeric mice were diluted 1:100 with PBS supplemented with 4% fetalbovine serum (FBS) after washing. The diluted sera were added 50μl/well, and then allowed to stand at 4° C. overnight. Aperoxidase-labeled anti-human IgM antibody (Biosorce, AHI0604) wasdiluted with PBS supplemented with 0.1% BSA after washing. Next thediluted chimeric mouse sera were added 50 μl/well and then allowed tostand overnight at room temperature. TMB was added to the product andreacted for 15 minutes. Then the reaction was stopped with 1N sulfuricacid. Absorbance at 450 nm was measured. Then an increase in theantibody titer was determined by reducing absorbance of a control well.FIG. 51 shows the results concerning anti-ASIALO GM1 human Igμ in seraof the chimeric mice CLH13-18; FIG. 52 shows the results concerninganti-GM2 human Igμ in sera of the chimeric mice CLH13-10. AS shown inthese figures, immunization with ganglioside resulted in the increasedhuman Igμ antibody titer specific to the antigen. That is, immuneresponse against the sugar chain antigen was confirmed in the humanantibody-producing chimeric mice of the present invention.

Three weeks after the third immunization, re-immunization was performedfor boosting. Three days after final immunization, the spleen wasexcised from the chimeric mouse, followed by cell fusion with PEGaccording to Example 24, thereby producing hybridomas. The humanantibodies against the ganglioside GM2 or ASIALO-GM1 obtained in thismanner are useful for treatment of HIV or cancer, such as melanoma.

EXAMPLE 85 Production of Mouse Strains Mainly Producing the CompleteHuman Antibody by Mating (B)

A male or female mouse, which retains SC20 fragment, and is homo- orheterozygous for heavy-chain-deficiency, and homo or heterozygous for κchain deficiency as produced according to Example 73, was mated with amale or female mouse, which retains W23 fragment, and is homo orheterozygous for heavy-chain deficiency, and homo- or heterozygous for κchain-deficiency as produced according to Example 73. This mating isexpected to yield progeny mice (double Tc/KO), which can meet fourconditions of retaining SC20 fragment and W23 fragment, and beinghomozygous for heavy-chain-deficiency and for κ chain-deficiency.

(1) Analysis of Single Tc/KO Mice

First, the following four types of analysis (A) to (D) were performedfor a total of 189 offspring produced by the mating above. A search wasperformed for a mouse (single Tc/KO), which meets only two of the aboveconditions (retaining no W23 fragment, and being heterozygous or wildtype for κ chain-deficiency). In these individuals, B lymphocytes weredeveloped by function of human heavy-chain introduced instead of mouseheavy-chain that had been deleted. In addition, most of the antibodymolecules in sera were thought to contain human heavy-chain.

(A) PCR was performed for DNA prepared from individual mouse to studythe retention of SC20 fragment in the same manner as in Example 68-(4).SC20 fragment-specific D14S543 was used as a primer.(B) Southern blotting was performed to study endogenous heavy-chaindeficiency in the same manner as in Example 49.(C) PCR was performed for DNA prepared from individual mouse to studythe retention of W23 fragment in the same manner as in Example 1. W23fragment-specific D2S1331 (Tomizuka et al., Nature Genetics, 1997,supra) was used as a primer.(D) Southern blotting was performed to study endogenous κ-chaindeficiency in the same manner as in Example 58.

Twenty out of 189 offspring were determined as single Tc/KO mice by thisanalysis.

50 μl of blood was collected from the single Tc/KO mouse HK83 (5 monthsold) and then 2 μl of 0.5 M EDTA (pH 8.0) was added to prevent the bloodfrom clotting. The blood sample was transferred into a centrifugationtube. Then 400 μl of sterile water was added to the tube. After 30seconds, 2×PBS containing 10% fetal calf serum (FCS) was added to thesolution, followed by centrifugation using a SEROMATIC-2 (KA2200,Kubota). After centrifugation, the supernatant was removed, and then theprecipitate was suspended in the remaining small amount of solution.Staining Medium (SM: 1 mM EDTA, 5% FCS, PBS containing 0.05% sodiumazide) was added to the suspension, followed by recentrifugation. Theprecipitate was suspended in approximately 10 μl of the remaining SMafter removal of the supernatant, 1 μl of Fc-block (Pharmingen, 01241A,anti-mouse CD32/CD16) was added to the suspension, and then the productwas allowed to stand on ice for 1 hour. After 1 hour, 1 μl of eachPE-labeled anti-mouse CD45R/B220 (Pharmingen, 01125A) and FITC-labeledanti-human Igm (Pharmingen, 08074D) was added, and then the product wasallowed to stand for 1 hour. Thus stained cells were washed twice withSM as described above. Finally the cells were suspended in 200 μl of SM,thereby preparing stained samples of peripheral blood nuclear cells.Flow cytometry analysis was made using a FACS-vantage (BectonDickinson). FIG. 53 shows the results. B220 positive cells, that is, Blymphocytes were absent in mouse antibody heavy-chain-deficient (KO)mice (ΔH/ΔH in the figure); but in single Tc/KO mice (ΔH/ΔH, SC20 in thefigure), the presence of a cell population of B lymphocytes which werepositive for both B220 and human p chain (hp in the figure). That is, itwas shown in single Tc/KO mice that introduction of SC20 fragmentrescued the deficiency of B lymphocytes.

Blood was collected from 8 single Tc/KO mouse individuals, HK7 (18week-old), HK38 (13 week-old), HK45 (13 week-old), HK47 (13 week-old),HK50 (13 week-old), HK61 (13 week-old), HK78 (17 week-old), and HK83 (17week-old). The concentration of human μ chain and of human γ chain insera were measured by ELISA as described in Example 14. Mouse μ chainwas detected by the method shown in Example 75 and mouse γ chain by thefollowing method for the sera collected as control from four individuals(litters born from the same mother as that of single Tc/KO mice; 13 to18 week-old) that were heterozygous for antibody heavy-chain deficiencyand heterozygous or wild type for antibody κ chain deficiency(containing no human chromosomal fragment). The concentration of mouse γchain was determined by ELISA according to Example 14. Anti-mouseimmunoglobulin γ chain antibodies (Sigma, 14280) diluted with PBS werecoated over a 96-well microtiter plate. Then the serum samples dilutedwith PBS containing fetal bovine serum (FBS), and peroxidase-labeledanti-mouse immunoglobulin γ chain antibodies (Caltag, M30107) were addedin order to incubate. The plate was washed, and then TMBZ (SUMITOMOBAKELITE COMPANY LIMITED, ML-1120T) was added as a peroxidase substrate,so that enzyme activity was evaluated with an absorbance at 450 nm. Theresults were compared with mouse immunoglobulin IgG (Pharmingen,03171D), having a γ chain with a known concentration in its purifiedstate and sequentially diluted with PBS containing FBS, so as todetermine the concentration of mouse immunoglobulin in sera. FIG. 54shows the results. The mean value of the measurements for each Ig isalso shown in this figure. The mean concentration of human μ chain insingle Tc/KO individuals was larger than that of mouse μ chain inantibody heavy-chain-deficient heterozygous individuals. Similarly, theconcentration of mouse μ chain and of mouse γ chain were measured forsingle Tc/KO individuals. No mouse μ chain was detected (1 μg/ml orless) in all the single Tc/KO mices; the mean concentration of mouse ychain was approximately one tenth of that of human γ chain. Theseresults suggested that in sera of single Tc/KO mice, most of antibodymolecules contained human heavy-chain.

The concentration of human γ chain subclass in sera was measured for theserum samples from four out of 7 said single Tc/KO mice by ELISA asdescribed in Example 29. FIG. 55 shows the results. Four types of hγsubclass were detected in all single Tc/KO mice.

0.5 ml of adjuvant (TiterMaxGold, Cytex corporation, G-1) was mixed with0.5 ml of human serum albumin (HSA, Sigma, A3782) which had beendissolved at the concentration of 0.25 mg/ml in PBS. Two single Tc/KOmice, HK45 (5 months old) and HK108 (3.5 months old), were immunizedsubcutaneously with 0.1 ml of the mixture. Blood was collected fromthese mice on day 0, 8, and 15 after immunization. The anti-HSA-human γchain antibody titer in sera was measured by ELISA as described inExample 21. On day 15 after immunization of the two single Tc/KO mice(immunized with HSA), a significant increase in the anti-HSA-human γchain antibody titer was observed. Thus, it was shown that in the singleTc/KO mouse, antigen specific antibodies (in this case, consisting ofhuman γ chain and mouse κ chain or mouse λ chain) were produced againstproteins derived from human for immunization.

(2) Analysis of Double Tc Mice

The following four types (A) to (D) of analysis were made for a total of189 offspring born. A search was done for mouse mice (double Tc), whichmeet at least

conditions and express human heavy-chain and human κ chainsimultaneously.

-   (A) To study the retention of SC20 fragment, PCR analysis was    performed for DNA prepared from the individual mice with the same    method as shown in Example 68-(4). The primer used herein was    SC20-specific D14S543.-   (B) To study the retention of W23 fragment, PCR analysis was    performed for DNA prepared from the individual mice with the same    method as shown in Example 68-(4). The primer used herein was W23    fragment-specific D2S1331 (Tomizuka et al., Nature Genetics, 1997,    supra).-   (C) Blood was collected from 3 to 6 week-old mice to confirm the    expression of human μ chain, and ELISA was performed according to    the method shown in Example 68-(4).-   (D) Blood was collected from 3 to 6 week-old mice to confirm the    expression of human κ chain, and ELISA was performed according to    the method shown in Example 20.

Twenty out of 189 mice were positive in all of the four types ofanalysis, and determined to be double Tc mice. Hence, it was confirmedthat SC20 and W23 fragments were simultaneously retained in anindividual mouse and their functions were expressed simultaneously.

(3) Analysis of Double Tc/KO Mice

DNAs of 20 double Tc mice were subjected to Southern blotting asdescribed in Examples 49 and 58 in order to analyze the state of mouseheavy-chain gene and mouse κ chain gene deficiency. The results were 6mice homozygous for both antibody heavy-chain-deficiency and κ chaindeficiencies. Thus 6 mice were homozygous for mouse heavy-chain and κchain deficiencies and determined to be double Tc/KO mice.

The peripheral blood nuclear cells prepared as described in Example85-(1) from two double Tc/KO mice, HKD5 (9 week-old) and HK190 (6week-old), were subjected to flow cytometry analysis according toExample 85-(1). Thus, in the two mice, the presence of B lymphocyteswhich were positive for B220 and for Hp was confirmed.

Blood was collected from three double Tc/KO mice, HK77 (17 week-old),HKD4 (6 week-old), and HKD5 (6 week-old) and the concentrations of humanμ chain and of human γ chain in sera were measured by ELISA as describedin Example 14. The mean concentration of human μ chain in sera and ofhuman γ chain in sera from the 3 mice were 360 mg/l and 201 mg/l,respectively. Both the concentrations of human μ chain and of human γchain were almost equivalent to that of single Tc/Ko mice shown in FIG.54. Furthermore, for HK190 (4 week-old) and HK192 (4 week-old) inaddition to the above 3 mice, the concentration of human κ chain in serawas detected as described in Example 20 and the concentration of mouse λchain was detected in the method as described below. The concentrationof mouse λ chain was detected by ELISA according to Example 14.Anti-mouse immunoglobulin λ chain antibodies (Caltag, M33600) dilutedwith PBS were coated over a 96-well microtiter plate, serum samplesdiluted with PBS containing fetal bovine serum (FBS), and thenperoxidase-labeled anti-mouse immunoglobulin λ chain antibodies (Caltag,M33607), were added to the plate for incubation. The plate was washed,and then TMBZ (SUMITOMO BAKELITE COMPANY LIMITED, ML-1120T) was added asa peroxidase substrate to evaluate the enzyme activity with absorbanceat 450 nm. The results were compared with that of IgG (Sigma, M6034),having a λ chain with a known concentration in its purified state andsequentially diluted with PBS containing FBS, so as to determine theconcentration of mouse immunoglobulin λ chain in sera. Based on theresults, first a proportion of the concentration of human κ chain to theconcentration of all light-chain (Human κ chain concentration+mouse λchain concentration) was obtained for individual mice. The mean value of5 individuals was 60%. Therefore, it was shown that the complete humanantibody molecule (human μ chain/κchain or human γ chain/κ chain)expressed dominantly over the hybrid antibody molecule (human μchain/mouse λ chain or human γ chain/mouse λ chain) in sera of thesedouble Tc/KO mice.

Blood was collected from double Tc/KO mice HKD5 (6 week-old) and theconcentration of hγ subclass in sera was measured by ELISA, similar toExample 85—(1). The results were human γ1 chain: 141 mg/l, human γ2chain: 61 mg/l, human γ3 chain: 119 mg/l, and human γ4 chain: 8.3 mg/l.All four types of human γ chain subclass were detected.

0.5 ml of adjuvant (TiterMaxGold, Cytex corporation, G-1) was mixed with0.5 ml of human serum albumin (HSA, Sigma, A3782) dissolved at aconcentration of 0.25 mg/ml in PBS. Two double Tc/KO mice, HK77 (20week-old) and HKD5 (10 week-old), were immunized subcutaneously with 0.1ml of the mixture. Blood was collected from these mice on day 0, 8, and15 after immunization. The anti-HSA-human γ chain antibody titer in serawas measured by a method similar to that of Example 21, and theanti-HSA-human κ chain antibody titer was measured by ELISA as describedin Example 23. The results are shown in FIGS. 56 and 57. On day 15 afterimmunization of the two double Tc/KO mice (immunized with HSA), asignificant increase was observed in both the anti-HSA-human γ chainantibody titer and the anti-HSA-human κ chain antibody titer. Thus, itwas shown that in a double Tc/KO mouse, the antigen specific completehuman antibody (in this case, containing human γ chain and κ chain) wasproduced against the proteins derived from human for immunization.

(4) Analysis of Double Tc/KO Mice (λ1 Mutant)

The mouse λ chain gene was not disrupted in the double Tc/KO mouse(Example 85-1(3)). Thus mouse λ chain protein was still expressed insera and the concentration thereof was approximately 40% of the whole Iglight-chain. To express human κ chain more efficiently, it is requiredto inactivate the competitive mouse λ chain gene.

The presence of mutant mice wherein expression of mouse λ chain isapproximately 1/50^(th) of that of normal mice is known (Ju et al., J.Immunol., 136: 2684, 1986). Gene analysis of this mouse strain revealedthat this was caused by the presence of base substitution in an Igλ1chain gene constant region (Ju et al., supra). In fact, almost no λ1chain accounting for 80% or more of the concentration of the whole λchain (remaining λ chains are λ2 chain and λ3 chain) was detected insera of homozygous mutant mice (herein after referred to as λ1 mutant)(Ju et al., supra). Hence, it is concluded that λ1 chain, the mainconstituent of mouse Igλ chain, is inactivated in the λ1 mutant.

Double Tc/KO mice (λ1 mutant) can be obtained by mating λ1 mutant withdouble Tc/KO mice described in Example 85-(3). This mouse will showdecreased expression of λ chain in serum and increased expression ofhuman κ chain instead. λ1 mutants were isolated from ICR mice (purchasedfrom CHARLES RIVER JAPAN, INC.) population by the above-mentioned method(Ju et al., supra). The isolated λ1 mutants were mated with double Tc/KOmice, thereby obtaining double Tc/KO (λ1 mutant) mice. Concentration ofhuman κ chain and of mouse λ chain in sera of the resultant double Tc/KO(λ1 mutant) and of double Tc/KO (λ1 mutant hetero or wild type) as acontrol were measured (Example 85-(3)). Based on these measurements,first, the percentage by human κ chain concentration of wholelight-chain concentration was determined for each mouse. Next, the meanpercentage of 5 mice was calculated for the two types of the mousestrains above. The results were 93% for double Tc/KO (λ1 mutant) and 63%for double Tc/KO (λ1 mutant hetero or wild type) mice. Furthermore, themean value of the whole light-chain concentration of 5 mice of eachstrain was 558 mg/l for double Tc/KO (λ1 mutant) and 525 mg/l for doubleTc/Ko (λ1 mutant hetero or wild type). That is, no significantdifference was found between the two.

These results suggested that the expression level of mouse λ chaindecreased in double Tc/KO (λ1 mutant), but that of human κ chainincreased as if it compensated for the decreased level. Therefore, theuse of the λ1 mutant was shown to enable efficient expression ofcomplete human antibody molecules consisting of human κ chain and humanheavy-chain.

EXAMPLE 86 Preparation of a HSA-Specific Human Antibody-ProducingHybridoma from the Double Tc/KO Mice

An anti-HSA human antibody-producing hybridoma was obtained from doubleTc/KO mice HKD5, for which the antibody titer had increased against HSAimmunization in Example 85. On day 30 after the initial immunization,final immunization was performed. 3 days later, hybridomas were producedby the method described in Example 24. The supernatants of approximately3300 wells, in which G418-resistant colonies had appeared, were analyzedby ELISA (Examples 14 and 61). 11 wells were positive for HSA-specifichuman μ chain; 39 wells were positive for HSA-specific human γ chain. 14wells out of 39 wells positive for anti-HSA human γ chain were positivefor human κ chain, and the remainder was positive for mouse λ chain. Inaddition, no well was positive for both types of Ig light-chain at thesame time. These results suggested that the hybridoma producingHSA-specific complete human antibodies (consisting of γ heavy-chain andκ light-chain) was obtained from the double Tc/KO mice. Moreover, ELISAanalysis was performed to detect four types of human γ chain subclassaccording to Example 61-(4). 7 wells were positive for γ1, 2 wellspositive for γ2, and 5 wells positive for γ4. Three typical clones ofIgG/κ hybridoma were subjected to limited dilution for subcloning, andthen the supernatant was used for measuring affinity. Measurement of theaffinity constant using surface plasmon resonance in BIAcore followed bycalculation using an attached calculation software resulted in 1.1×10¹⁰to 6.6×10¹⁰ M⁻¹. Thus, the anti-HSA human IgG/κ antibody obtained fromHKD5 was shown to have high affinity to HSA.

EXAMPLE 87 Construction of Cassette Vectors ploxPHyg and ploxPbsr

In the following Examples 87 to 103, production of human artificialchromosomes λ-HAC and κ-HAC, having human antibody λ light-chain andhuman antibody κ light-chain gene clusters cloned thereto, will bedescribed. In addition, introduction of each of the produced HAC into amouse, expression of a human antibody gene contained in HAC in a mouse,and transfer of HAC to chimera mouse offspring. The outline of thesystem for producing HAC-introduced mouse disclosed herein is shown inFIGS. 58 and 59.

Cassette vectors ploxPHyg and ploxPbsr were constructed as follows forinsertion of a loxP sequence, a recognition sequence of Cre recombinantenzyme, onto a human chromosome. For positive selection of cellscarrying translocation as expected, the two cassette vectors wereconstructed such that the former contained PGK promoter and the lattercontained GFP gene to be transcribed by this PGK promoter.

First, the construction of ploxPHyg will be described. A plasmidpBluescript II SK(−) (TOYOBO) was cleaved with a restriction enzymeEcoRV (Boehringer). The product was allowed to react for 30 minutes at50° C. using dephosphorylase CIAP (alkaline dephosphorylase derived fromthe small intestine of a calf, TAKARA SHUZO CO., LTD.), therebydephosphorylating the cleaved ends. DNA fragments were excised from aplasmid pBS302 (GIBCO) using restriction enzymes SpeI and HindIII(Boehringer) and then blunt-ended with a DNA blunting kit (TAKARA SHUZOCO., LTD.). Then the DNA fragment was ligated using a DNA Ligation kit(TAKARA SHUZO CO., LTD.), so that a competent cell DH5 (TOYOBO) wastransformed, thereby obtaining a plasmid pBS302HS. Blunt-ending,ligation and transformation were conducted following protocols attachedto each kit.

Next, the plasmid was cleaved with a restriction enzyme SalI(Boehringer) and then dephosphorylated. To this plasmid, PGK promoterfragments excised from a plasmid pGKPuro (WHITEHEAD INSTITUTE,distributed by Dr. Peter W. Laird) with restriction enzymes SalI andXhoI (Boehringer) were cloned, thereby obtaining a plasmid pBSPGK302HS.The plasmid was cleaved with restriction enzymes EcoRI and NotI(Boehringer), blunt-ended, and dephosphorylated. Then a NotI linker(TAKARA SHUZO CO., LTD.) was ligated to the plasmid, thereby obtaining aplasmid pBSPGK302HSN.

On the other hand, a hyglomycin B-resistant gene cassette was excisedfrom a plasmid #1-133 (distributed by Shunichi Takeda, Professor,Medical School, Kyoto University) with a restriction enzyme BamHI(Boehringer), and then the ends were blunted. The plasmid pBSPGK302HSNwas cleaved with a restriction enzyme SalI (Boehringer) and blunt-ended.Next the hygromycin B-resistant gene cassette was cloned, therebyobtaining a plasmid ploxPHyg.

Next, construction of ploxPbsr will be described. First, SfiI linker wasinserted into the SacI site of a plasmid pBluescript II SK(−) (TOYOBO)as described above. The Sfi linker had been prepared by synthesizingoligo DNA with the following sequence and phosphorylating the 5′ end(synthesized by GREINER JAPAN).

(Sfi linker) 5′-GGCCGC [A/T]GCGGCC-3′ (SEQ ID NO: 75)

The plasmid was cleaved with a restriction enzyme BamHI (Boehringer),and dephosphorylated. Then a DNA fragment excised from a plasmid pBS302(GIBCO) with a restriction enzyme BamHI (Boehringer) was cloned, therebyobtaining a plasmid pBSSfK302B. A SpeI linker (TAKARA SHUZO CO., LTD.)was inserted into the ClaI site of a plasmid PGREEN LANTERN-1 (GIBCO) asdescribed above. A GFP gene cassette fragment excised from a restrictionenzyme SpeI (Boehringer) was cloned into the plasmid pBSSfK302B that hadbeen cleaved with SpeI followed by dephosphorylation, thereby obtaininga plasmid pBSSfK302BGFP. This plasmid was cleaved with a restrictionenzyme XbaI (Boehringer) and blunt-ended. Then a DNA fragment(blastcidin S-resistant gene cassette) that had been cleaved with arestriction enzyme BamHI (Boehringer) from a plasmid #1-134 (distributedby Shunichi Takeda, Professor, Medical School, Kyoto University) andblunt-ended was cloned, thereby obtaining ploxPbsr. FIG. 60 shows thestructure of the cassette vectors ploxPHyg and of ploxPbsr.

EXAMPLE 88 Construction of Targeting Vectors pHCF2loxPHyg(F) and (R)

Targeting vectors pHCF2loxPHyg(F) and (R) were constructed as followsfor insertion of a loxP sequence into a HCF2 locus (closely linked toIgλ region in the centromere side) on human chromosome #22. Forward (F)and reverse (R) targeting vectors were constructed because thetranscriptional direction of HCF2 gene was unknown (whether it is fromcentromere to telomere or from telomere to centromere). First, thegenomic region of the human HCF2 locus was amplified by PCR using thefollowing primers:

(SEQ ID NO: 76) HCF2-F2K; 5′-TCGAGGTACCGTGAGAACAAGACAGAGAATGAGGGAGG-3′(SEQ ID NO: 77) HCF2-R2K; 5′-TCGAGGTACCTAATGCAGAGGCTCTTTGGTGTACTTGG-3′PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,LA Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec and 68° C. for 15 minutes. PCR products were treated with proteinaseK (GIBCO) and then subjected to gel filtration with a CHROMA SPIN-TE400(CLONTECH). Then the product was cleaved with a restriction enzyme KpnI(Boehringer) and subjected to gel filtration with a CHROMA SPIN-TE1000(CLONTECH). The resulting PCR fragment was cloned into the KpnI site ofa plasmid pBluescriptII, thereby obtaining pHCF2. Next the DNA fragmentcontaining a loxP was, cleaved with restriction enzymes KpnI and NotI(Boehringer) from a cassette vector ploxPHyg, blunt-ended, and thencloned into the SnaBI site of the plasmid pHCF2. The product wherein thedirection of the loxP sequence was same as that of the cloned HCF2 genefragment was designated as (F), and the product with that in theopposite direction was designated as (R) [pHCF2loxPHyg(F) and (R), FIGS.61 and 62].

EXAMPLE 89 Construction of a Targeting Vector pRNR2loxPbsr

A targeting vector pRNR2loxPbsr was constructed as follows for insertionof a loxP sequence into a RNR2 locus on a human chromosome #14. First,the genomic region of the human RNR2 locus was amplified by PCR usingthe following primers:

(SEQ ID NO: 78) RNR2-F10E; 5′-TCGAGAATTCAGTAGCTGGCACTATCTTTTTGGCCATC-3′(SEQ ID NO: 79) RNR2-R10E; 5′-TCGAGAATTCGGAGAAAGAACACACAAGGACTCGGTC-3′PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,LA Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, and 68° C. for 15 min. PCR products were treated with proteinase K(GIBCO) and then subjected to gel filtration with a CHROMA SPIN-TE400(CLONTECH). Then the product was cleaved with a restriction enzyme EcoRI(Boehringer) and then subjected to a CHROMA SPIN-TE1000 (CLONTEC). Onthe other hand, the KpnI site of a plasmid pBluescriptII was deleted bycleaving the site with a restriction enzyme KpnI (Boehringer), bluntingand self-ligation. Then a SrfI linker was inserted into the NotI site toconstruct a plasmid vector [pBS(K)Sr]. In addition, oligo DNA with thefollowing sequence was used as the SrfI linker.

SrfI linker; 5′-GCCCGGGC-3′ (SEQ ID NO: 80)Next a plasmid pRNR2 was constructed by cloning the PCR fragment of theabove RNR2 gene into the EcoRI site of the plasmid [pBS(K)Sr].

Moreover, a KpnI linker (TAKARA SHUZO CO., LTD.) was inserted into theSfiI site of the cassette vector ploxPbsr, a DNA fragment containing aloxP sequence was cleaved with a restriction enzyme KpnI (Boehringer),then the fragment was cloned into the KpnI site of the plasmid pRNR2.The RNR2 gene was reported to be transcribed in the direction fromtelomere to centromere (R. G. Worton et al., SCIENCE, 239:64-68, 1988).Thus, the product wherein the direction of the cloned PCR fragment ofthe RNR2 gene was opposite to that of the loxP sequence was used as atargeting vector (pRNR2loxPbsr, FIG. 63).

EXAMPLE 90 Construction of a Targeting Vector pYHZloxPHyg

A targeting vector pYHZloxPHyg was constructed as follows for insertionof a loxP sequence into the genomic region (located approximately 30 kbaway from the Igλ region in the direction of the centromere side)cosYHZ304 (obtained from Shimizu, Professor, School of Medicine, KeioUniversity) on human chromosome #2. First, the human cosYHZ304 genomicregion was amplified by PCR using the following primers:

(SEQ ID NO: 81) YHZ-F2B; 5′-TCGAGGATCCGATAGAGAGATTGTCTTAAATGGGTGGG-3′(SEQ ID NO: 82) YHZ-R2B; 5′-TCGAGGATCCAACAGCTGGAACTCATAAAAGCATAGC-3′PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,LA Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, and 68° C. for 15 min. PCR products were treated with proteinase K(GIBCO) and then subjected to gel filtration with a CHROMA SPIN-TE400(CLONTECH). Then the product was cleaved with a restriction enzyme BamHI(Boehringer) and subjected to gel filtration using a CHROMA SPIN-TE1000(CLONTEC). Moreover, the NotI site of the plasmid pBluescriptII wasdeleted by cleaving the site with a restriction enzyme NotI(Boehringer), blunting and self-ligation. Then a SrfI linker wasinserted into the SacII site, so as to construct a plasmid vector[PBS(N)Sr]. A plasmid pYHZ was constructed by cloning the PCR fragmentof the above cosYHZ304 genomic region into the BamHI site of the plasmid[pBS (N) Sr]. The plasmid pYHZ was cleaved with a restriction enzymeTth111I (TAKARA SHUZO CO., LTD.) and blunted. Then a NotI linker (TAKARASHUZO CO., LTD.) was inserted into the product, thereby obtaining PYHZN.In addition, Furthermore, a NotI linker (TAKARA SHUZO CO., LTD.) wasinserted into the KpnI site of the cassette vector ploxPHyg, a DNAfragment containing a loxP sequence was cleaved with a restrictionenzyme NotI (Boehringer), and then the resultant fragment was clonedinto the NotI site of the plasmid pYHZN. The direction of the cosYHZ304sequence is known to be from telomere to centromere according toShimizu, Professor, School of Medicine, Keio University. Thus, theproduct wherein the direction of the cloned PCR fragment of thecosYHZ304 genome was opposite to that of the loxP sequence was used as atargeting vector (pYHZloxPHyg, FIG. 64).

EXAMPLE 91 Construction of a Cassette Vector pTELPuro

A cassette vector pTELPuro was constructed as follows for insertion of ahuman telomere sequence onto a human chromosome. The human telomeresequence was synthesized by PCR according to J. J. Harrington et al.(Nature Genet., 15, 345-355, 1997) and then cloned into the EcoRV siteof a plasmid pBluescript II SK(−) (TOYOBO), thereby obtaining a plasmidpTEL. After the EcoRI site of a plasmid pGKPuro (distributed byWHITEHEAD INSTITUTE, Dr. Peter W. Laird) was changed to the NotI site,the DNA fragment (puromycin-resistant gene cassette) cleaved with arestriction enzyme NotI (Boehringer) was cloned into the NotI site ofthe plasmid pTEL, thereby obtaining a cassette vector pTELPuro (FIG.65).

EXAMPLE 92 Construction of Targeting Vectors pTELPuroCDBA(F) and (R)

Targeting vectors pTELPuroCDBA (F) and (R) were constructed as followsfor insertion of a human telomere sequence into a CD8A locus (closelylinked to the Igκ region in the telomere side) on human chromosome #2.Forward (F) and reverse (R) targeting vectors were constructed becausethe transcriptional direction of the CD8A gene had been unknown (whetherit is from centromere to telomere or from telomere to centromere).First, the genomic region of the human CD8A locus was amplified by PCRusing the following primers:

(SEQ ID NO: 83) CD8A-F; 5′-TCGAGGATCCCTTTAGTGAAGGCAAAGGAAGGGACACTC-3′(SEQ ID NO: 84) CD8A-R; 5′-TCGAGGATCCTGTAAAGGGGTAGCCTGTCCTCTTTCATG-3′PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,LA Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, and 65° C. for 10 min. PCR products were treated with proteinase K(GIBCO) and then subjected to gel filtration with a CHROMA SPIN-TE400(CLONTECH). Then the product was cleaved with a restriction enzyme BamHI(Boehringer) and subjected to gel filtration with a CHROMA SPIN-TE1000(CLONTECH). The resulting PCR fragment was cloned into the BamHI site ofthe plasmid pTELPuro. Next the cloned CD8A genomic fragment, whosedirection was same as that of the human telomere sequence, wasdetermined as (F), the fragment whose direction was opposite to thecloned CD8A genomic fragment as (R) [pTELPuroCD8A(F) and (R), FIGS. 66and 67].

EXAMPLE 93 Site-Directed Insertion of a loxPHyg Cassette onto HumanChromosome #22 in a Chicken DT-40 Cell

Insertion of the loxPHyg cassette to the HCF2 locus was attempted bytransfecting the targeting vectors pHCF2loxPHyg(F) and (R) constructedas in Example 88 into a chicken DT-40 cell retaining human chromosome#22 fragment in which telomere-directed truncation occurred at the LIFlocus (Kuroiwa et al., Nucleic Acid Research, 26: 3447-3448, 1998).

Chicken DT-40 cells were cultured in a RPMI1640 medium (GIBCO)supplemented with 10% fetal bovine serum (GIBCO, herein after referredto as FBS), 1% chicken serum (GIBCO), and 10⁻⁴M2-mercaptoethanol(Sigma). Approximately 10⁷ cells were washed once with an additive-freeRPMI1640 medium, and suspended in 0.5 ml of an additive-free RPMI1640medium. Twenty five to 30 μg of the targeting vectors pHCF2loxPHyg (F)and (R) linearized with a restriction enzyme NotI (Boehringer) wereadded to the suspension, and the mixture was transferred to a cuvettefor electroporation (Bio-Rad) and allowed to stand for 10 minutes atroom temperature. The cuvette was set in a gene pulsar (Bio-Rad), andwas impressed with voltage (550 V and 25 μF). The product was allowed tostand for 10 minutes at room temperature, and cultured for 24 hours.After 24 hours, the medium was exchanged with that containing hygromycinB (1 mg/ml). The product was dispensed into five 96 well culture plates,followed by selection culture for approximately 2 weeks. Genome DNAswere extracted from the hygromycin B-resistant clones using a PuregeneDNA Isolation Kit (Gentra System). Genome DNAs were cleaved withrestriction enzymes HpaI and XhoI (Boehringer), subjected toelectrophoresis in 0.8% agarose gel, followed by alkaline blotting ontoa cellulose nitrate filter (DuPont). Southern hybridization wasperformed on this filter using a HCF2 probe to identify the homologousrecombinant. The HCF2 probe was constructed by PCR using the followingprimers and a human genome as a template. A ³²P-labeled DNA probe wasconstructed by random priming using a PCR product as a template(Amersham, following the attached protocol).

HCF2-F4; 5′-CACATGACAAGAGCTCAGCG-3′ (SEQ ID NO: 85) HCF2-R4;5′-TCTGACTTCCTCATGAGAGCC-3′ (SEQ ID NO: 86)PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,Ex Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. Each cycle consisted of 1 min, 98° C. for 10 sec,65° C. for 30 sec, and 72° C. for 1 min. For the targeting vectorpHCF2loxPHyg(F), a band not less than 20 kb band was detected for thenon homologous recombinant, and a 8.7 kb band for the homologousrecombinant. For the targeting vector pHCF2loxPHyg(R), a band not lessthan 20 kb band was detected for the non homologous recombinant, and a9.5 kb band for the homologous recombinant (FIGS. 61 and 62). Southernhybridization results showed that 41 out of 52 clones were the targethomologous recombinants in the targeting vector pHCF2loxPHyg(F), and 28out of 60 clones were the target homologous recombinants in (R) (calledHF and HR clone, respectively).

EXAMPLE 94 Site-Directed Insertion of a loxPbsr Cassette onto HumanChromosome #14 in a Chicken DT-40 Cell

Insertion of the loxPbsr cassette to the RNR2 locus was attempted bytransfecting the targeting vector pRNR2loxPbsr constructed as in Example89 into a chicken DT-40 cell retaining fragment SC20 of a humanchromosome #14 fragment SC20 (Kuroiwa et al., Nucleic Acid Research, 26:3447, 1998; distributed by Shunichi Takeda, Professor, Medical School,Kyoto University).

As described above, the targeting vector pRNR2loxPbsr linearized with arestriction enzyme SrfI (TOYOBO) was transfected, followed by selectionculture for about 2 weeks under the presence of blastcidin S (10 μg/ml).Genome DNAs were extracted from the resistant clones, and then thehomologous recombinants were identified by PCR using the followingprimers (FIG. 63).

(SEQ ID NO:87) RNR2-1; 5′-TGGATGTATCCTGTCAAGAGACC-3′ (SEQ ID NO:88)STOP-3; 5′-CAGACACTCTATGCCTGTGTGG-3′

PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,LA Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec. and 65° C. for 5 min. Hence, approximately 2.5 kb of a PCR productwas amplified as expected in 8 out of 60 clones. That is, the homologousrecombinant was obtained (called R clone).

EXAMPLE 95 Site-Directed Cleavage of Human Chromosome #2 in a ChickenDT-40 Cell

Cleavage of chromosome #2 at an insertion site was attempted bytransfecting the targeting vectors pTELPuroCD8A(F) and (R) constructedas in Example 92 into a chicken DT-40 cell retaining the full lengthhuman chromosome #2 (Kuroiwa et al., Nucleic Acid Research, 26: 3447,1998; distributed by Shunichi Takeda, Professor, Medical School, KyotoUniversity) so as to insert a human telomere sequence to the CD8A locus.

As described above, the targeting vectors pTELPuroCD8A(F) and (R)linearized with a restriction enzyme SrfI (TOYOBO) were transfected,followed by selection culture for about 2 weeks in the presence ofpuromycin (0.3 μg/ml). Genome DNAs were extracted from the resistantclones, and then the homologous recombinants were identified by PCRusing the following primers (FIGS. 66 and 67).

For the targeting vector pTELPuroCD8A(F) CD8A-3;5′-GCCCTCATGGAAATCTCCTGGG-3′ (SEQ ID NO: 89) CDPuro-1;5′-GCAGCAACAGATGGAAGGCCTC-3′ (SEQ ID NO: 90) For the targeting vectorpTELPuroCD8A(R) CD8A-2; 5′-GAACAGAAAGCCACTCTTGCTTTCCAT-3′ (SEQ ID NO:91) CDPuro-2; 5′-ACCGAGCTGCAAGAACTCTTCCTCAC-3′ (SEQ ID NO: 92)PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,LA Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, and 68° C. for 10 min. Hence, PCR products were amplified asexpected in 2 out of 95 clones for (F); in 2 out of 160 clones for (R).That is, the homologous recombinants were obtained (called CD clone andCR clone, respectively).

Next, PCR and FISH analysis were performed to confirm cleavage ofchromosome #2 occurred at the CD8A locus in the homologous recombinants,CD and CR clones.

(1) PCR Analysis

The presence of the gene and polymorphic markers on chromosome #2 (FIG.68) was detected by PCR using the following primers. Primers used fordetection of D2S373, D2S113, D2S388, D2S1331, D2S134, D2S171, and TPOwere manufactured by BIOS.

Vκ3 detection primers Vκ3-F; 5′-CTCTCCTGCAGGGCCAGTCA-3′ (SEQ ID NO: 93)Vκ3-R; 5′-TGCTGATGGTGAGAGTGAACTC-3′ (SEQ ID NO: 94) Cκ detection primersCκ-F; 5′-TGGAAGGTGGATAACGCCCT-3′ (SEQ ID NO: 95) Cκ-R;5′-TCATTCTCCTCCAACATTAGCA-3′ (SEQ ID NO: 96)PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,Ex Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, and 56-60° C. for 30 sec, and 72° C. for 30 sec. FIG. 68 shows theresult. All markers were detected in the chicken DT-40 cell clones 521D4retaining the full length human chromosome #2, whereas all the markerslocated on the telomere side beyond CD8A disappeared in the homologousrecombinant CD clone. Furthermore in the homologous recombinant CRclone, all markers were detected.

(2) FISH Analysis

To visually determine that human chromosome #2 was cleaved at the CD8Alocus, FISH was performed using a pGKPuro probe capable of detecting apuromycin-resistant gene in a targeting vector (FIG. 69), according toKuroiwa et al's method (Nucleic Acid Research, 26: 3447-3448, 1998).COT1 staining (labeled with rhodamin, red) revealed the chromosome #2was fragmented in the CD clone (CD10) compared to 521D4. Furthermore,the signal (labeled with FITC, yellow) derived from the pGKPuro probewas detected on the one end of the telomere, suggesting that the CD8Alocus, at which the targeting vector had been inserted, is the one endof the telomere of the chromosome #2 fragment. In the CR clone, thesignal derived from the pGKPuro probe was detected at 2p12, suggestingthat no fragmentation occurred.

In conclusion, the human chromosome #2 was cleaved at the CD8A locus inthe homologous recombinant CD clone. In addition, no fragmentation inthe CR clone suggests that the transcriptional direction of the CD8Agene is from centromere to telomere. As described above,telomere-directed truncation in either direction can be applied todetermine an unknown transcriptional direction for a locus.

EXAMPLE 96 Site-Directed Insertion of a loxPHyg Cassette onto HumanChromosome #2 in a Chicken DT-40 Cell

Insertion of the loxPHyg cassette at the cosYHZ304 genomic region wasattempted by transfecting the targeting vector pYHZloxPHyg constructedas in Example 90 into the chicken DT-40 cell CD clone retaining thehuman chromosome #2 fragment in which telomere-directed truncationoccurred at the CD8A locus obtained in Example 95 above.

As described above, the targeting vector pYHZloxPHyg linearized with arestriction enzyme SrfI (TOYOBO) was transfected, followed by selectionculture for about 2 weeks in the presence of hygromycin B (1 mg/ml).Genome DNAs were extracted from the resistant clones, and then thehomologous recombinants were identified by PCR using the followingprimers (FIG. 64).

YHZ-2; 5′-TCCTCTTTTTCCTTCCTTTGCCTC-3′ (SEQ ID NO: 97) Yhyg-2;5′-ATTATTTTGGGCGTTGCGTGG-3′ (SEQ ID NO: 98)PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,LA Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98 for 10 sec,and 65° C. for 5 ml. The homologous recombinant (called Y clone) can beidentified by this analysis.

EXAMPLE 97 Construction of a Human Artificial λ-HAC Having both a HumanAntibody Heavy-Chain Gene Cluster and a λ Light-Chain Gene Cluster

To express a complete human antibody stably and efficiently in a mouseas described in the general description, construction of a humanartificial chromosome. λ-HAC having both a human antibody heavy-chaingene cluster and a λ light-chain gene cluster was attempted bytranslocating of a human chromosome #22 fragment consisting ofHCF2-Igλ-LIF, to the RNR2 locus on a human chromosome #14 fragment SC20containing human IgH.

First, a DT40 hybrid cell retaining both the human chromosome #22fragment and the chromosome #14 fragment SC20 was constructed by cellfusion of the homologous recombinants HF and HR clones obtained inExamples 93 and 94 above with the homologous recombinant R clone.

(1) Construction of a DT40 Hybrid Cell Retaining Both the HumanChromosome #22 Fragment and the Chromosome #14 Fragment SC20.

The R clone was cultured in a RPMI1640 medium containing blastcidin S(10 μg/ml) and HF clone in the same medium containing hygromycin B (1mg/ml). The two types of clones (1-2×10⁷ clones) were mixed, subjectedto centrifugation, and washed twice with a serum-free RPMI1640 medium.After complete removal of the remaining medium, 0.5 ml of 50% PEG 1500(Boehringer) kept at 37° C. was added gently to the product, followed byvigorous mixing for approximately 2 minutes with a pipette. To themixture, 1 ml of a serum-free RPMI1640 medium was added slowly for 1minute, and then 9 ml of a serum-free RPMI1640 medium was added slowlyfor 3 minutes. Then the mixture was allowed to stand for 10 minutes at37° C. Subsequently, the mixture was centrifuged at 1200 rpm for 5minutes, and cultured in an RPMI1640 medium containing serum for 24 to48 hours. Then the medium was replaced by an RPMI1640 medium containingblastcidin S (10 μg/ml) and hygromycin B (1 mg/ml), dispensed into five24-well culture plates, and then cultured for 3 to 4 weeks. Similarly,the HR and R clones were fused (cell fusion). Genome DNAs were extractedfrom a hybrid cell (called RHF clone) obtained from cell fusion of theHF and R clones and a hybrid cell (called RHR clone) obtained from theHR and R clones. PCR was performed using the following primers,confirming retention of two chromosomes, human chromosomes 14 and 22.

Human chromosome #14 detection primers VH3-F; 5′-AGTGAGATAAGCAGTGGATG-3′(SEQ ID NO: 99) VH3-R; 5′-GTTGTGCTACTCCCATCACT-3′ (SEQ ID NO: 100) Humanchromosome #22 detection primers Igλ-F; 5′-GAGAGTTGCAGAAGGGGTGACT-3′(SEQ ID NO: 101) Igλ-R; 5′-GGAGACCACCAAACCCTCCAAA-3′ (SEQ ID NO: 102)PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,Ex Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, 56° C. for 30 sec, and 72° C. for 30 sec. PCR showed that 6 clonesof the RHF clones and 2 clones of the RHR clones were positive for bothVH3 and Igλ. In addition, FISH using a human COT1 DNA as a proberevealed that all of these clones retained two individual humanchromosomes. The above results suggest that the RHF and RHR hybridclones retain two chromosomes, human chromosomes 14 and 22.

(2) Site-Directed Translocation of the Human Chromosome #22 Fragment tothe Chromosome #14 Fragment SC20 in the RHF and RHR Hybrid Clones

(2)-1 Construction of a Vector pBS185hisd Stably Expressing CreRecombinant Enzyme

As described in the general description, site-directed translocation ofa human chromosome is performed using the Cre-loxP system. With thissystem, the recombination efficiency between non homologous chromosomesis expected to be very low. Thus the following expression vector wasconstructed to express Cre enzyme stably, not transiently.

The Cre recombinant enzyme expression vector pBS185 (GIBCO) was cleavedwith a restriction enzyme EcoRI (Boehringer), followed by insertion of aBglII linker, thereby obtaining pBS185Bg. A histidinol-resistant genecassette was excised from a plasmid #1-132 (distributed by ShunichiTakeda, Professor, School of Medicine, Kyoto University) with arestriction enzyme BamHI (Boehringer), and then cloned into the BglIIsite of the pBS185Bg, thereby obtaining a vector pBS185hisD (FIG. 70).

(2)-2 Site-Directed Translocation of the Human Chromosome #22 Fragmentto the Chromosome #14 Fragment SC20 in the RHF and RHR Hybrid ClonesUsing the Cre-loxP System.

As described above, the Cre recombinant enzyme stable expression vectorpBS185hisD linearized with a restriction enzyme KpnI (Boehringer) wasseparately transfected to the RHF and RHR hybrid clones, and subjectedto selection culture for approximately 2 weeks in the presence ofhistidinol (0.5 mg/ml). Subsequently, the resistant cell population wasdispensed into four 6-well culture plates (24 pools). Two pools wererandomly selected and cultured to approximately 10⁷ cells. The cell poolwas suspended in 4 ml of PBS (phosphoric acid buffer) supplemented with5% FBS and 1 μg/ml propydium iodide(PI), and subjected to analysis withFACSVantage (Becton Dickinson). As described above, since recombination(translocation) between loxPs results in reconstruction and expressionof GFP gene, cells carrying translocation can be detected by FACS.Sorting was repeated 4 times for the cell fraction suspected to bepositive for GFP. Culture was conducted after every sorting in aRPMI1640 medium containing hygromycin B (1 mg/ml) in order to remove thecells retaining acentric chromosomes as described below. As a result,GFP-positive cells could be concentrated with a purity of 98-99% fromthe RHF clone, however, none could be concentrated from the RHR clone.It was suggested that the directions of two loxPs were the same as eachother in the RHF clone (from centromere to telomere) as shown in FIG. 71and that translocation resulted in a normal chromosomal structure.However, in the RHR clone, the directions of two loxPs differed fromeach other so that translocation resulted in a dicentric chromosomehaving two centromeres and an acentric chromosome lacking a centromere.For example, this can cause no growth in the presence of hygromycin B.In other words, no cells can be concentrated. The results show thespecificity of the experiment for translocation with this system. Inaddition, HCF2 gene is expected to be transcribed in the direction fromcentromere to telomere.

Next, whether recombination occurred between loxPs as expected wasconfirmed by PCR for the two clones (called SF2-21 and SF2-23) cloned byFACS from RHF. Genome DNAs were extracted from the SF2-21 and SF2-23 andPCR was performed using the following primers.

PGK-1; 5′-ATAGCAGCTTTGCTCCTTCG-3′ (SEQ ID NO: 103) GFP-1;5′-TTCTCTCCTGCACATAGCCC-3′ (SEQ ID NO: 104)

PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,EX Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, 61° C. for 30 sec and 72° C. for 1 min. As shown in FIG. 72, whenthe PCR with these primers yields a product of about 600 bp, thissuggests that recombination occurred between loxPs. FIG. 73 shows theresult. In the RHF clone to which no Cre recombinant enzyme stableexpression vector was transfected, no PCR product was obtained, whereasin SF2-21 and SF2-23, a PCR product of approximately 600 bp wasobtained.

In addition, FISH was performed using a human 14q ter-specific probe(the long arm telomere region of human chromosome #14 was detected,FITC-labeled) and a pGKPuro probe (the long arm telomere region of humanchromosome #22 fragment was detected, Rhodamin-labeled). Thus, FITC andrhodamin signals were detected in the telomere regions on both ends ofthe same chromosome (FIG. 74). Another FISH using a human chromosome#14-specific probe (labeled with rhodamin) and human chromosome#22-specific probe (labeled with FITC) also resulted in detection ofsignals derived from the both probes on the same chromosome (FIG. 74).

In conclusion, translocation occurred in SF2-21 and SF2-23 as expectedand a human artificial chromosome λ-HAC containing both the humanheavy-chain gene cluster and the λ light-chain gene cluster on the samechromosome was constructed.

EXAMPLE 98 Construction of a Human Artificial Chromosome κ-HACContaining Both Human Antibody Heavy-Chain and κ Light-Chain GeneClusters

A human artificial chromosome κ-HAC containing both human antibodyheavy-chain and κ light-chain gene clusters can be constructed bytranslocation of the human chromosome #2 fragment consisting ofcosYHZ304-Igκ-CD8A to the RNR2 locus on the chromosome #14 fragment SC20containing human IgH.

First, a DT40 hybrid cell retaining both the human chromosome #2fragment and the chromosome #14 fragment SC20 was constructed by cellfusion of the homologous recombinant Y clone obtained in Example 96above with the homologous recombinant R clone.

(1) Construction of a DT40 Hybrid Cell Retaining Both the HumanChromosome #2 Fragment and the Chromosome #14 Fragment SC20.

The R clone was cultured in a RPMI1640 medium containing blastcidin S(10 μg/ml) and the Y clone in the same medium containing hygromycin B (1mg/ml). The two types of clones (1-2×10⁷ clones) were mixed, subjectedto centrifugation, and washed twice with a serum-free RPMI1640 medium.After complete removal of the remaining medium, 0.5 ml of 50% PEG 1500(Boehringer) kept at 37° C. was added gently to the product, followed byvigorous mixing for approximately 2 minutes with a pipette. To themixture, 1 ml of a serum-free RPMI1640 medium was added slowly for 1minute, and then 9 ml of a serum-free RPMI1640 medium was added slowlyfor 3 minutes. Then the mixture was allowed to stand for 10 minutes at37° C. Subsequently, the mixture was centrifuged at 1200 rpm for 5minutes, and cultured in a RPMI1640 medium containing serum for 24 to 48hours. Then the medium was replaced by a RPMI1640 medium containingblastcidin S (10 μg/ml) and hygromycin B (1 mg/ml), dispensed into five24-well culture plates, and then cultured for 3 to 4 weeks. Genome DNAswere extracted from the hybrid clones (called RY clone), and subjectedto PCR using the following primers, thereby confirming the retention oftwo chromosomes, human chromosomes 14 and 2.

Human chromosome #14 detection primers VH3-F; 5′-AGTGAGATAAGCAGTGGATG-3′(SEQ ID NO: 105) VH3-R; 5′-GTTGTGCTACTCCCATCACT-3′ (SEQ ID NO: 106)Human chromosome #2 detection primers Cκ-F; 5′-TGGAAGGTGGATAACGCCCT-3′(SEQ ID NO: 107) Cκ-R; 5′-TCATTCTCCTCCAACATTAGCA-3′ (SEQ ID NO: 108)PCR was performed using GeneAmp9600 (Perkin-Elmer) as a thermal cycler,Ex Taq (TAKARA SHUZO CO., LTD.) as a Taq polymerase, and the attachedbuffer and dNTP (dATP, dCTP, dGTP and dTTP) following the recommendedconditions. PCR was performed for 35 cycles following thermaldenaturation at 94° C. for 1 min. Each cycle consisted of 98° C. for 10sec, 56-60° C. for 30 sec, and 72° C. for 30 sec. Furthermore, FISH wasperformed using human COT1 DNA as a probe to confirm that the two humanchromosomes exist independently. The above results can confirm that theRY hybrid clone retains two chromosomes, human chromosomes 14 and 2.

(2) Site-Directed Translocation of the Human Chromosome #2 Fragment tothe Chromosome #14 Fragment SC20 in the RY Hybrid Clone

As described above, the Cre recombinant enzyme stable expression vectorpBS185hisD linearized with a restriction enzyme KpnI (Boehringer) wastransfected to the RY hybrid clone, and subjected to selection culturefor approximately 2 weeks in the presence of histidinol (0.5 mg/ml).Subsequently, the resistant cell population was dispensed into four6-well culture plates (24 pools). Two pools were randomly selected andcultured to approximately 10⁷ cells. The cell pool was suspended in 4 mlof PBS (phosphoric acid buffer) supplemented with 5% FBS and 1 μg/mlpropydium iodide (PI), and subjected to analysis with FACSVantage(Becton Dickinson). Sorting was repeated several times for the cellfraction suspected to be positive for GFP.

Similarly, whether recombination occurs as expected between loxPs inRY-derived clones to be cloned by FACS can be confirmed by PCR usingPGK-1 and GFP-1 primers. This can also be confirmed by FISH using ahuman 14q ter-specific probe (long arm telomere region of the humanchromosome #14 is detected, FITC-labeled) and a pGKPuro probe (short armtelomere region of the human chromosome #2 fragment is detected,Rhodamin-labeled). Furthermore, FISH using a human chromosome#14-specific probe and a human chromosome #2-specific probe can alsoreach a conclusion that a human artificial chromosome κ-HAC containingboth the human heavy-chain gene cluster and the κ light-chain genecluster on the same chromosome is constructed.

EXAMPLE 99 Introduction of Human Artificial Chromosomes λ-HAC and K-HACfrom a DT40 Hybrid Cell into a Chinese Hamster CHO Cell

λ-HAC and κ-HAC were first introduced into a CHO cell by MMCT for thepurpose of introducing λ-HAC and κ-HAC into a mouse ES cell.

SF2-21 and SF2-23 DT40 hybrid cells were separately cultured on 8 Petridishes with a diameter of 150 mm. When the cells reached a confluentstate, the medium was exchanged with a RPMI1640 medium supplemented with20% FBS, 1% chicken serum, 10⁴M2-mercaptoethanol, 0.05 μg/ml colcemid,and cultured for another 36 hours to form a microcell. The cells weresuspended in 24 ml of a serum-containing RPMI1640 medium, and dispensed2 ml into each of twelve 25 cm² centrifugation flasks precoated with 100μg/ml poly L-lysin. Then the cells were cultured for 1 hour at 37° C.allowing the cells to adhere to the bottom of the flasks. Followingremoval of the culture solution, cytochalasin B (10 μg/ml, Sigma)solution kept at 37° C. was added to a centrifugation flask, thensubjected to centrifugation at 8000 rpm for 1 hour at 34° C. Themicrocell was suspended in a serum-free DMEM medium, and purified with a8 μm and 5 μm filters. Following purification, the product wascentrifuged at 1700 rpm, for 10 minutes, and then suspended in 5 ml of aserum-free DMEM medium. On the other hand, approximately 10⁷ CHO cellswere removed by trypsinization. The cells were washed twice in aserum-free DMEM medium, and then suspended in 5 ml of a serum-free DMEMmedium. Again, the microcell was centrifuged at 1700 rpm for 10 minutes.Without removing the supernatant, 5 ml of the CHO suspension was layeredgently over the supernatant. Following centrifugation, the culturesolution was removed, 0.5 ml of 1:1.4 PEG solution [5 g of PEG1000 (WakoPure Chemicals Co., Ltd) and 1 ml of DMSO (Sigma) were dissolved in 6 mlof DMEM] was added, followed by vigorous stirring with a pipette forapproximately 2 minutes. Subsequently, 10 ml of a serum-free DMEM mediumwas added gently to the mixture for about 3 minutes, and allowed tostand for 10 minutes at 37° C. After centrifugation, the cells weresuspended in a F12 medium (GIBCO) supplemented with 10% FBS, dispensedinto ten 24-well culture plates, and cultured for 24 hours at 37° C.Then the medium was exchanged with a F12 medium containing 800 μg/mlG418, followed by selection culture for 3 to 4 weeks.

Genome DNAs were extracted from the G418-resistant clones. Then the CHOclone retaining λ-HAC was identified by PCR in the conditions asdescribed above using Igλ and VH3 detection primers, PGK-1, and GFP-1primers (data not shown). Moreover, FISH was performed using humanchromosome #14-specific and 22-specific probes for the clone positive inthe PCR above, thereby visually confirming the presence of λ-HAC. Theseresults suggested that a CHO clone retaining λ-HAC was obtained.

In exactly the same manner, CHO cells retaining κ-HAC can be cloned.

EXAMPLE 100 Introduction of λ and κ-HAC from a CHO Cell into a Mouse ESCell

To produce a chimeric mouse retaining λ and κ-HAC, λ and κ-HAC retainedin a CHO cell were introduced to a mouse ES cell by MMCT.

According to Tomizuka et al's method (Nature Genet. 16: 133, 1997), amicrocell was purified from the CHO cell retaining λ and κ-HAC ofapproximately 10⁸, then suspended in 5 ml of DMEM. Approximately 10⁷mouse ES cells, TT2F, were removed by trypsin treatment, washed threetimes with DMEM, and suspended in 5 ml of DMEM. Next the suspension wasadded to the centrifuged microcell, and subjected to centrifugation at1250 rpm for 10 minutes so as to completely remove the supernatant. Theprecipitate was mixed well by tapping. Then 0.5 ml of 1:1.4 PEG solution[5 g of PEG 1000 (Wako Pure Chemicals Co., Ltd) and 1 ml of DMSO (Sigma)were dissolved in 6 ml of DMEM], followed by stirring well forapproximately 1.5 minutes. Subsequently 10 ml of DMEM was slowly addedto the product. Then the product was centrifuged for 10 minutes at 1250rpm, and suspended in 30 ml of an ES medium. The suspension wasdispensed into 3 Petri dishes (CORNING) with a diameter of 100 mmcontaining feeder cells pre-inoculated therein for culturing. 24 hourslater, the medium was exchanged with a medium containing 300 μg/ml G418,followed by selection culture for approximately 1 week. Genome DNAs wereextracted from the drug-resistant colonies, and then PCR was performedin conditions as described above using Igλ or Cκ and VH3 detectionprimers. Furthermore, FISH was performed using probes specific to humanchromosomes 14 and 22, or 2. In conclusion, the ES cell clone retainingthe target human artificial chromosomes λ and κ-HAC was obtained.

EXAMPLE 101 Production of a Chimeric Mouse Retaining Human ArtificialChromosomes λ and λ-HAC

A chimeric mouse was produced in the manner as described in Example 10using the ES cell clone as obtained in Example 100 above. Possible hostsinclude ICR and MCH (CLEA JAPAN, INC.) or an embryo at the 8-cell cellstage obtained by mating antibody heavy-chain knockout female and malemice as established in Example 67-(1). The coat color of the progenymice born as a result of transplantation of the injected embryo intosurrogate mice determines whether they are chimeric or not (Example68-(3)). Chimeric mice can be obtained from the ES cell line retaininghuman artificial chromosome λ-HAC or that retaining κ-HAC. It issuggested that the ES cell line retaining human artificial chromosomeλ-HAC or that retaining κ-HAC possesses chimera-forming ability, thatis, retaining ability to differentiate into the normal tissue of amouse.

EXAMPLE 102 Expression of a Complete Human Antibody in a Chimeric MouseRetaining Human Artificial Chromosomes λ-HAC and κ-HAC

Retention of HAC by chimeric mice is as shown by PCR and FISH analysis(Examples 97 and 98) and the method (Tomizuka et al., Nature Genetics,16, 133-143). The expression of complete human antibody moleculesconsisting of human Igλ chain, human Ig heavy-chain and human Igλchain/heavy-chain in a chimeric mouse retaining λ-HAC is confirmed bythe method described in Example 65. Moreover, the expression of completehuman antibody molecules consisting of human Igκ chain, human Igheavy-chain and human Igκ chain/heavy chain in a chimeric mouseretaining κ-HAC is also confirmed by the method described in Example 65.

EXAMPLE 103 Transfer of Human Artificial Chromosomes λ-HAC and κ-HACfrom the Chimeric Mice to its Progeny

Transfer of λ-HAC and κ-HAC from the chimeric mice to its progeny wasexamined by the method described in Example 68-(4). As a result, the twomouse strains, retaining and transferring λ-HAC and κ-HAC, respectivelyto its progeny are established. The retention of HAC in these micestrains is shown by PCR and FISH analysis (Examples 97 and 98) and themethod (Tomizuka et al., Nature Genetics, 16, 133-143). In addition, theexpression of complete human antibody molecules consisting of human Igλchain, human Ig heavy-chain and human Igλ chain/heavy-chain in the mousestrain retaining and transferring λ-HAC to its progeny is confirmed bythe method as described in Example 65. The expression of complete humanantibody molecules consisting of human Igκ chain, human Ig heavy-chainand human Igκ chain/heavy-chain in the mouse strain retaining andtransferring κ-HAC to its progeny is confirmed by the method asdescribed in Example 65. As described in Example 73, the mouse strainretaining and transferring λ-HAC or κ-HAC to its progeny is repeatedlymated with a mouse strain deficient in endogenous antibody heavy-chainand light-chain κ genes, whereby the mouse strains retaining λ-HAC orκ-HAC and being homozygous for endogenous antibody heavy-chain gene andκ chain gene deficiency can be obtained. Complete human antibodies aremainly produced in these mouse strains.

Stable retention of each HAC is examined for the mouse strains retainingand transferring λ-HAC or κ-HAC to its progeny by the method describedin Example 68-(5). The results show the stable retention of each HAC inthe somatic cells of the mouse strains.

EXAMPLE 104 Introduction of the Human Chromosome #22 Fragment in whichTelomere-Directed Truncation Occurred at LIF Locus into a ChineseHamster CHO Cell

The human chromosome #22 fragment in which telomere-directed truncationoccurred at LIF locus was introduced into CHO cells by MMCT forintroduction of the fragment into mouse ES cells.

The chicken DT-40 cell clones retaining the human chromosome #22fragment in which telomere-directed truncation had occurred at LIF locuswere separately cultured on 8 Petri dishes with a diameter of 150 mm.When the cells reached a confluent state, the medium was exchanged byRPMI1640 media supplemented with 20% FBS, 1% chicken serum,10⁻⁴M2-mercaptoethanol, and 0.05 μg/ml colcemid, and cultured foranother 36 hours to form the microcell. The cells were suspended in 24ml of a serum-containing RPMI1640 medium, and dispensed 2 ml into eachof twelve 25 cm² centrifugation flasks (CORNING) precoated with 100μg/ml poly L-lysin. Then the cells were cultured for 1 hour at 37° C.for the cells to adhere to the bottom of the flask. Following removal ofthe culture solution, a centrifugation flask was filled withcytochalasin B (10 μg/ml, Sigma) solution kept at 37° C., and thensubjected to centrifugation at 8000 rpm for 1 hour at 34° C. Themicrocells were suspended in serum-free DMEM media, and purified with a8 μm and 5 μm filters. Following purification, the product wascentrifuged at 1700 rpm, for 10 minutes, and then suspended in 5 ml of aserum-free DMEM medium. On the other hand, approximately 10⁷ CHO cellswere removed by trypsinization. The cells were washed twice in aserum-free DMEM medium, and then suspended in 5 ml of a serum-free DMEMmedium. Again, the microcell was centrifuged at 1700 rpm for 10 minutes.Without removing the supernatant, 5 ml of the previously prepared CHOsuspension was layered gently. Following centrifugation, the culturesolution was removed, 0.5 ml of 1:1.4 PEG solution [5 g of PEG1000 (WakoPure Chemicals Co., Ltd) and 1 ml of DMSO (Sigma) were dissolved in 6 mlof DMEM], followed by vigorous stirring with a pipette for approximately2 minutes. Subsequently, 10 ml of a serum-free DMEM medium was addedgently to the mixture for about 3 minutes, and allowed to stand for 10minutes at 37° C. After centrifugation, the cells were suspended in aF12 medium (GIBCO) supplemented with 10% FBS, dispensed into ten 24-wellculture plates, and cultured for 24 hours at 37° C. Then the medium wasexchanged with a F12 medium containing 800 μg/ml G418, followed byselection culture for 3 to 4 weeks.

Genome DNAs were extracted from the G418-resistant clones and subjectedto PCR using Igλ, D22S315, D22S272, D22S278 detection primers and Puro-1and LIF-1 primers (Kuroiwa et al., Nucleic Acid Research, 26:3447-3448,1998). Thus, two markers, D22S272 and D22S278 were not detected asexpected, however, the other markers were all detected. In addition,FISH using human COT1 DNA and pGKPuro probes revealed that two humanchromosome #22 fragments were retained and a signal derived from pGKPuroprobe was observed on each of the one end of the telomere. Accordingly,it was concluded that the CHO cell clone retaining two individual humanchromosome #22 fragments in which telomere-directed truncation hadoccurred at LIF locus was obtained.

EXAMPLE 105 Introduction of Human Chromosome #22 Fragment C68 into anEndogenous Antibody Heavy-Chain and κ Chain Deficient Mouse ES CellRetaining Human Chromosome #14 Fragment SC20

The human chromosome #22 fragment C68 obtained in Example 83 wasintroduced by the microcell method into the mouse ES cell line HKD2-1 asproduced by the method in Example 61-(1) to be deficient in endogenousantibody heavy-chain and κ chain and to retain the human chromosome #14fragment SC20. Except the use of the CHO cell clone C68-6 retaining twohuman chromosome #22 fragments obtained in Example 104 as chromosomedonor cells, the method described in Example 35 was employed. Theresultant puromycin- and G418-resistant strain (double resistant strain)NLH (CHO1) was subjected to PCR analysis, confirming the retention ofthe introduced chromosomal fragments. Three types of primers, D14S543(Example 68) for the chromosome #14 fragment, and Igλ and D22S315 forchromosome #22 (Example 2) were used in total. Thus, all three types ofthe markers were confirmed to be present in the NLH(CH01) strain. Inaddition, FISH analysis was performed using a human chromosome-specificprobe (Example 68). Microscopic study of 50 nuclear plates showed thattwo individual chromosomal fragments hybridizing to the probes wereobserved for 45 (90%) nuclear plates. These two chromosomal fragmentswere thought to be human chromosome #14 fragment SC20 and humanchromosome #22 fragment C68, respectively. Therefore, NLH(CH01) strainwas confirmed to retain the chromosome #14 fragment SC20 and thechromosome #22 fragment C68 simultaneously.

EXAMPLE 106 Detection and Quantification of Human Antibodies in Sera ofthe Chimeric Mice Produced by Injecting Endogenous Antibody Heavy-Chainand κ Chain Gene Deficient Mouse ES Cells Retaining the Human Chromosome#14 Fragment SC20 and the Human Chromosome #22 Fragment C68 intoImmunodeficient Mouse Host Embryos

A chimeric mouse was produced by the method shown in Example 10 and thelike from the mouse ES cell line NLH(CH01) obtained in Example 105. Thehost embryo used herein was an embryo at the 8-cell stage obtained bymating male and female antibody heavy-chain knockout mice established inExample 67-(1). A total of 307 injected embryos were transplanted sothat 32 progeny mice were born. Whether progeny are chimeric or not wasdetermined by their coat color. That is, if progeny exhibits wild typecolor (dark brown) among white coat color derived from the host embryo(ICR), it is determined as a chimeric mouse. Fourteen out of 32 progenymice born clearly exhibited wild type coat color, for which thecontribution of the ES cells was confirmed. These results confirms thatthe endogenous antibody heavy-chain gene- and κ chain gene-deficientmouse ES cell line NLH(CH01), retaining human chromosome #14 fragmentSC20 and human chromosome #22 fragment C68 simultaneously, retainschimera-forming ability, that is, retains the ability to differentiateinto normal mouse tissue.

Blood was collected from 6 to 10 week-old chimeric mice derived fromNLH(CH01), and subjected to ELISA according to Examples 14, 32 and 85,for quantification of various human immunoglobulin concentrations andmouse immunoglobulin λ chain concentration in sera. Table 29 shows theresult. High concentrations of human μ chain, γ chain, and λ chain weredetected in sera of the chimeric mice. The mouse λ chain carryingundisrupted genes was also detected, however, its concentration waslower than that of human λ chain.

These results suggested that the expression of complete human antibodymolecules consisting of human heavy-chain and human λ chain in thechimeric mouse was dominant over that of hybrid antibody moleculesconsisting of human heavy-chain and mouse λ chain. That is, it wasconfirmed that the human chromosome introduced via the chicken DT-40cell functions in the mouse and protein encoded by the human gene on thehuman chromosome is expressed.

Transfer of fragment C68 from the chimeric mice to its progeny mice wasexamined by the method described in Example 68-(4). Thus a mouse strainretaining fragment C68 and transferring it to progeny was established.The retention of fragment C68 in the mouse strain is shown by PCR andFISH analysis (Example 105). Furthermore, a mouse strain (C68+SC20)retaining both fragments SC20 and C68 can be obtained by mating thismouse with the mouse strain retaining and transferring chromosome #14fragment SC20 to its progeny. The expression of complete human antibodymolecules consisting of human Igλ chain, human Ig heavy-chain and humanIgλ chain/heavy-chain in the mouse strain (C68+SC20) is confirmed by themethod described in Example 65.

TABLE 29 Human μ Human γ Human λ Mouse λ Mouse chain chain chain chainname (mg/l) (mg/l) (mg/l) (mg/l) C-1 840 90 1300 150 C-3 560 170 470 170C-12 980 650 1700 440

EXAMPLE 107 Preparation of a HSA-Specific Human Antibody-ProducingHybridoma from the Chimeric Mice Produced by Injecting EndogenousAntibody Heavy-Chain and κ Chain Gene-Deficient Mouse ES Cells, whichRetain Human Chromosome #14 Fragment SC20 and Human Chromosome #22Fragment C68, into Immunodeficient Mouse Host Embryos.

The chimeric mice C-10 (derived from NLH(CH01); 30% chimerism) asproduced in Example 106 were immunized with HSA. A HSA solution 0.25mg/ml was prepared by mixing human serum albumin (HSA, Sigma, A3782)dissolved in PBS and an adjuvant (TiterMaxGold, Cytrx). The mouse wasimmunized twice with 0.15 ml of the HSA solution (0.05 ml each wasinjected subcutaneously at three positions in total). Initialimmunization was performed on the 7 week-old mice, and 21 days later, asecond immunization was performed. According to Example 61 or 66,anti-HSA antibody concentration in sera diluted 1:100 was measured. FIG.75 shows the results. Since an increase in anti-HSA human antibodyconcentration was confirmed, final immunization was performed on day 30with HSA dissolved in PBS. One clone producing HSA-specific human μchain and human λ chain was obtained by the production and cloning ofhybridomas according to Example 66.

These results show that human Igλ chain expressed by the humanchromosome introduced via the chicken DT-40 cell binds specifically toHSA and is functional.

EXAMPLE 108 Retention of the Introduced Human Chromosomal Fragments bythe Somatic Cells of the Chimeric Mice Produced by Injecting EndogenousAntibody Heavy-Chain and κ Chain Gene-Deficient Mouse ES Cells, whichRetain Human Chromosome #14 Fragment and Human Chromosome #22 Fragment,into Immunodeficient Mouse Host Embryos

Fibroblasts derived from the tails of the chimeric mice C-12 (derivedfrom NLH(CH01); chimerism 99%) produced in Example 106 were culturedfollowing the method (Tomizuka et al., Nature Genetics, 16, 133-143).Preparation of Chromosome Samples from the Fibroblasts and FISH analysiswere performed according to the method (Tomizuka, et al., supra).Microscopic examination for 50 nuclear plates revealed that 21 (42%)nuclear plates contained two independent chromosomes hybridizing tohuman COT1 probes. These two chromosomes are thought to be chromosome#14 fragment SC20 and chromosome #22 fragment C68, respectively. Hence,it was shown that the somatic cell of the chimeric mouse retains twotypes of chromosomal fragments.

EXAMPLE 109 Preparation of a Complete Human Antibody-Producing HybridomaAgainst Sugar Chain Antigen (GM2) from the Chimeric Mice as Produced byInjecting Endogenous Antibody Heavy-Chain and Light-Chain-DeficientMouse ES Cells, which Retains Human Chromosome #14 Partial Fragment andHuman Chromosome #22 Partial Fragment, into Immunodeficient Mouse HostEmbryos

The hybridomas as obtained in Example 84-(3) were subjected to selectionculture in the presence of G418 using 96 well plates. Then theantibodies in the culture solution were subjected to screening by ELISAaccording to Example 84-(3). Cloning was performed by the limiteddilution method, thereby obtaining 11 clones of the antibody-producinghybridoma. The antibodies in the culture supernatant were analyzed byELISA so that binding of the obtained monoclonal antibodies to GM2 wasconfirmed.

These results suggest that the complete human antibody-producinghybridoma against sugar chain antigen ganglioside GM2 can be obtainedfrom the chimeric mouse. The obtained human antibody is expected toapply for treating diseases such as HIV and melanoma.

EXAMPLE 110 Preparation of a Complete Human Antibody-Producing HybridomaAgainst Sugar Chain Antigen (Asialo-GM1) from the Endogenous AntibodyHeavy-Chain and Light-Chain-Deficient Mice Retaining Human Chromosome#14 Partial Fragment and Human Chromosome #2 Partial Fragment

The Tc mice HK232 as produced in Example 85 were immunized withasialo-GM1. An adjuvant (MPL+TDM Emulsion, RIBI Immunochem ResearchInc., R-700) was dissolved in 2 ml of a mixture of chloroform andmethanol (2:1). This solution was mixed with 1 mg of asialo-GM1 (ISOSEPAB, 65/12) dissolved in 2 ml of a mixture of chloroform and methanol(2:1). The mixture was transferred into a round-bottom flask, and driedwith a rotary evaporator. Two ml of PBS was added to the dried productand mixed vigorously using a Vortex mixer, thereby preparing suspensionGg4-RIBI. The mouse was immunized intraperitonealy with 0.2 ml ofGg4-RIBI and 500 μg of anti-mouse CD40 antibodies. One week after theimmunization, blood was collected, and an increase in the antibody titerwas confirmed by ELISA according to Example 84. Two weeks after theimmunization, final immunization was performed intraperitonealy with thesuspension Gg4-RIBI while 5 μg of recombinant human IL-6 wassubcutaneously injected. Three days after final immunization, the mousespleen was excised, followed by cell fusion according to Example 24.Thus the hybridomas were produced. Selection culture was performed inthe presence of G418, or G418 and puromycin using 96-well plates. Thenthe antibodies in the culture solution were subjected to screening byELISA according to Example 84. Moreover, 0.15 mg of asialo-GM1, 1.5 mgof Glactocerebroside, and 1.5 mg of cholesterol were dissolved in 2 mlof a mixture of chloroform and methanol (2:1) and dried. Next the driedproduct was resuspended in 100 ml of PBS, dispensed 100 μl per well intoELISA plates for solid phase formation. Screening was performed by ELISAusing the prepared plates. The cells in the wells shown as positive bythe two types of screening above were cloned. The culture supernatant ofthe anti-asialo-GM1 human IgM antibody-producing hybridomas were allowedto react with HIV-infected cells and analyzed by a flowcytometry.Therefore, one clone of the hybridoma producing monoclonal antibodiesthat bind to HIV-infected cells rather than HIV-uninfected cells wasobtained.

These results suggest that the complete human antibody-producinghybridoma against sugar chain antigen Asialo-GM1 is obtained from thedouble Tc mouse. Thus obtained human antibody is expected to apply fortreatment for diseases, such as HIV.

EXAMPLE 111 Preparation of a Complete Human Antibody-Producing HybridomaAgainst Human TNF-α from the Endogenous Antibody Heavy-Chain andLight-Chain-Deficient Mouse Retaining Human Chromosome #14 PartialFragment and Human Chromosome #2 Partial Fragment

Hybridomas were produced by immunizing the 8 week-old Tc mice HK492 asproduced in Example 85 with human TNF-α according to the methods ofExamples 84 and 85. TNF-α solution dissolved in PBS was mixed with thesame amount of adjuvant (Titer Max Gold, CytRx). The mice were immunizedwith this mixture 25 μg/mouse. Immunization was performed overapproximately 80 days at an interval of 2 to 3 weeks. After an increasein the antibody concentration was confirmed, final immunization wasperformed intraperitonealy with only TNF-α solution. Then hybridomaswere produced according to Example 86. The cells in the wells positivefor the antibody were cloned, so that one clone of the anti-TNF-α humanIgG/κ antibody-producing hybridoma was obtained.

All of the publications, patents, and patent applications cited in thisspecification are all incorporated as reference into this specification.

INDUSTRIAL APPLICABILITY

The present invention provides a chimeric non-human animal retainingsingle or multiple foreign chromosome(s) or fragment(s) thereof, andexpressing the gene on the chromosome(s) or on the fragment(s) thereof.Biologically active substances can be produced using the chimericnon-human animal of this invention.

The present invention provides a pluripotent cell retaining single ormultiple foreign chromosome(s) or fragment(s) thereof, and expressingthe gene on the chromosome(s) or on the fragments thereof. Using thiscell, hereditary diseases can be treated by bone marrow transplantationand the like.

The present invention provides a pluripotent cell carrying at least twotypes of disrupted endogenous genes. By using the cell of this inventionas a recipient cell, into which single or multiple foreign chromosomesor fragments thereof containing the same gene as the disruptedendogenous gene or a gene homologous to the same are introduced, afunctional cell or a chimeric non-human animal, which retains single ormultiple foreign chromosome(s) or fragment(s) thereof, and expresses thegene on the foreign chromosome(s) or on fragment(s) thereof, can beproduced. Expression of the gene on single or multiple foreignchromosome(s) or on fragment(s) thereof in such a chimeric non-humananimal or its progeny, its tissues or its cells thereof allowsproduction of a biologically active substance.

Furthermore, the present invention provides a method for artificiallymodifying a chromosome(s) or fragment(s) thereof.

Free Text of Sequence List

SEQ ID NOS:1-58 and 61-74 show nucleotide sequences of primers.

SEQ ID NOS:59 and 60 show nucleotide sequences of probes.

SEQ ID NO:75 shows a nucleotide sequence of Sfi I linker.

SEQ ID NOS:76-79 show nucleotide sequences of primers.

SEQ ID NO:80 shows a nucleotide sequence of Srf I linker.

SEQ ID NOS:81-108 show nucleotide sequences of primers.

1. A method for producing a chimeric non-human animal comprising amodified foreign chromosome(s) or a fragment(s) thereof, which comprisesthe steps of: (a) preparing a microcell comprising a foreignchromosome(s) or a fragment(s) thereof, and transferring said foreignchromosome(s) or a fragment(s) thereof into a cell with high homologousrecombination efficiency through its fusion with said microcell; (b) insaid cell with high homologous recombination efficiency, inserting avector by homologous recombination into a desired site of said foreignchromosome(s) or a fragment(s) thereof, and/or a desired site of achromosome(s) derived from said cell with high homologous recombinationefficiency, thereby marking said desired site; (c) in said cell withhigh homologous recombination efficiency, causing deletion and/ortranslocation to occur at the marked site of said foreign chromosome(s)or a fragment(s) thereof; and (d) preparing a microcell comprising saidforeign chromosome(s) or a fragment(s) thereof in which deletion ortranslocation has occurred, and transferring said foreign chromosome(s)or a fragment(s) thereof into a pluripotent non-human animal cellthrough its fusion with said microcell.
 2. The method of claim 1,wherein a plurality of said cells with high homologous recombinationefficiency are subjected to whole cell fusion after steps (a) and (b)and are subjected to step (c).
 3. The method of claim 2, wherein aplurality of said cells with high homologous recombination efficiencyeach comprise a distinct foreign chromosome(s) or a fragment(s) thereof.4. The method of claim 1, wherein said targeting vector comprises atelomere sequence which is introduced into a desired site by insertionof the targeting vector.
 5. The method of claim 4, wherein said deletionoccurs at a site where said telomere sequence has been introduced. 6.The method of claim 1, wherein said targeting vector comprises arecognition sequence for a site-directed recombination enzyme, and saidrecognition sequence is introduced into a desired site by insertion ofthe targeting vector.
 7. The method of claim 6, wherein a vector, whichis capable of expressing a site-directed recombination enzyme, isintroduced into said cell with high homologous recombination efficiencysimultaneously with or after insertion of said targeting vectorcomprising said recognition sequence for a site-directed recombinationenzyme, so that an activity of said site-directed recombination enzymeis expressed, resulting in deletion and/or translocation of said foreignchromosome(s) or a fragment(s) thereof at a site into which saidrecognition sequence is introduced.
 8. The method of claim 7, whereinsaid translocation occurs between a plurality of foreign chromosomes orfragments thereof.
 9. The method of claim 7, wherein said translocationoccurs between said foreign chromosome(s) or a fragment(s) thereof andsaid chromosome(s) derived from said cell with high homologousrecombination efficiency.
 10. The method of claim 6, wherein saidsite-directed recombination enzyme is a Cre enzyme.
 11. The method ofclaim 6, wherein said recognition sequence for site-directedrecombination enzyme is a LoxP sequence.
 12. The method of claim 1,wherein said cell with high homologous recombination efficiency is anembryonic stem cell (or ES cell).
 13. The method of claim 1, whichfurther comprises a step of screening cells comprising a foreignchromosome(s) or a fragment(s) thereof in which deletion and/ortranslocation has occurred.
 14. The method of claim 13, wherein saidscreening is based on expression of a marker gene.
 15. The method ofclaim 14, wherein said marker gene is a drug-resistance gene.
 16. Themethod of claim 14, the marker gene is a green fluorescentprotein-encoding gene derived from the jellyfish Aequorea victoria or amodified gene thereof.
 17. The method of claim 1, wherein in the step(d), a microcell is produced from said cell with high homologousrecombination efficiency; said foreign chromosome(s) or a fragment(s)thereof, in which deletion and/or translocation has occurred istransferred into a CHO cell through its fusion with said microcell; amicrocell is produced from the CHO cell; and then said foreignchromosome(s) or a fragment(s) thereof in which deletion and/ortranslocation has occurred is transferred into a pluripotent cellthrough its fusion with said microcell.
 18. The method of claim 1, saidpluripotent cell is an embryonic stem cell (or ES cell).
 19. The methodof claim 1, said foreign chromosome(s) or a fragment(s) thereof isderived from a human.
 20. A method for producing a non-human animalcomprising a modified foreign chromosome(s) or a fragment(s) thereof,which comprises the steps of: (a) preparing a microcell comprising aforeign chromosome(s) or a fragment(s) thereof, and transferring saidforeign chromosome(s) or a fragment(s) thereof into a cell with highhomologous recombination efficiency through its fusion with saidmicrocell; (b) in said cell with high homologous recombinationefficiency, inserting a vector by homologous recombination into adesired site of said foreign chromosome(s) or a fragment(s) thereof,and/or a desired site of a chromosome(s) derived from said cell withhigh homologous recombination efficiency, thereby marking said desiredsite; (c) in said cell with high homologous recombination efficiency,causing deletion and/or translocation to occur at the marked site ofsaid foreign chromosome(s) or a fragment(s) thereof; (d) preparing amicrocell comprising said foreign chromosome(s) or a fragment(s)thereof, in which deletion and/or translocation has occurred, andtransferring said foreign chromosome(s) or a fragment(s) thereof into acell derived from a non-human animal through its fusion with saidmicrocell; and (e) transplanting the nucleus of said cell derived fromthe non-human animal into an enucleated unfertilized egg derived from ahomologous non-human animal of the same species.
 21. The method of claim20, wherein a plurality of said cells with high homologous recombinationefficiency are subjected to whole cell fusion after steps (a) and (b)and are subjected to the step (c).
 22. The method of claim 21, wherein aplurality of said cells with high homologous recombination efficiencycomprise a distinct foreign chromosome(s) or a fragment(s) thereof. 23.The method of claim 20, wherein said targeting vector comprises atelomere sequence, which is introduced into a desired site by insertionof the targeting vector.
 24. The method of claim 23, wherein saiddeletion occurs at a site into which a telomere sequence has beenintroduced.
 25. The method of claim 20, wherein said targeting vectorcomprises a recognition sequence for site-directed recombination enzyme,and said recognition sequence is introduced into a desired site byinsertion of the targeting vector.
 26. The method of claim 25, wherein avector, which is capable of expressing a site-directed recombinationenzyme, is introduced into said cell with high homologous recombinationefficiency simultaneously with or after insertion of said targetingvector comprising said recognition sequence for a site-directedrecombination enzyme, so that an activity of said site-directedrecombination enzyme is expressed, resulting in deletion and/or atranslocation of said foreign chromosome(s) or fragment(s) thereof at asite into which said recognition sequence is introduced.
 27. The methodof claim 26, wherein said translocation occurs between a plurality offoreign chromosomes or fragments thereof.
 28. The method of claim 26,wherein said translocation occurs between said foreign chromosome(s) ora fragment(s) thereof and said chromosome derived from a cell with highhomologous recombination efficiency.
 29. The method of claim 25, whereinsaid site-directed recombination enzyme is a Cre enzyme.
 30. The methodof claim 25, wherein said recognition sequence for a site-directedrecombination enzyme is a LoxP sequence.
 31. The method of claim 20,wherein said cell with high homologous recombination efficiency is anembryonic stem cell (or ES cell).
 32. The method of claim 20, whichfurther comprises a step of screening cells containing a foreignchromosome(s) or a fragment(s) thereof in which deletion and/ortranslocation has occurred.
 33. The method of claim 32, wherein saidscreening is based on expression of a marker gene.
 34. The method ofclaim 33, wherein said marker gene is a drug-resistant gene.
 35. Themethod of claim 33, wherein said marker gene is a green fluorescentprotein-encoding gene derived from the jellyfish Aequorea victoria or amodified gene thereof.
 36. The method of claim 20, wherein, in the step(d), a microcell is produced from said cell with high homologousrecombination efficiency; said foreign chromosome(s) or fragment(s)thereof, in which deletion and/or translocation have/has occurred,is/are transferred into a CHO cell through its fusion with themicrocell; a microcell is produced from the CHO cell; and then saidforeign chromosome(s) or a fragment(s) thereof, in which deletion and/ortranslocation has occurred, is transferred into a cell derived from anon-human animal through its fusion with the microcell.
 37. The methodof claim 20, said cell derived from a non-human animal is a culture cellderived from an embryo or a blastocyst.
 38. The method of claim 20, saidcell derived from a non-human animal is a culture cell derived from afetus or an adult.
 39. The method of claim 20, said cell derived from anon-human animal is a fibroblast cell derived from fetus.
 40. The methodof claim 20, said foreign chromosome(s) or a fragment(s) thereof isderived from a human.
 41. A non-human animal, which retains achromosomal fragment(s) obtained by deletion of a foreign chromosome(s)or a fragment(s) thereof.
 42. The non-human animal of claim 41, whereinsaid chromosomal fragment(s) comprises: (i) a marker gene and telomeresequence, and/or (ii) a recognition sequence for a site directedrecombination enzyme.
 43. A non-human animal, comprising a recombinantforeign chromosome(s) obtained by translocation between a plurality offoreign chromosomes or fragments thereof.
 44. The non-human animal ofclaim 43, wherein said recombinant chromosomal fragment(s) comprises:(i) a marker gene and a telomere sequence; and/or (ii) a recognitionsequence for a site-directed recombination enzyme.
 45. The non-humananimal of claim 43, wherein said recombinant foreign chromosome(s) isindependently maintained in the nucleus of the non-human animal cell.46. The non-human animal of claim 43, wherein said recombinant foreignchromosome(s) is derived from a human.
 47. The non-human animal of claim43, wherein the recombinant foreign chromosome(s) is derived from humanchromosomes #14 and #2.
 48. The non-human animal of claim 43, whereinsaid recombinant foreign chromosome(s) is derived from human chromosomes#14 and #22.
 49. The non-human animal of claim 43, wherein saidrecombinant foreign chromosome(s) comprises genes for a heavy-chain anda light-chain λ of a human antibody.
 50. The non-human animal of claim43, wherein said recombinant foreign chromosome(s) comprises genes for aheavy-chain and a light-chain κ gene of a. human antibody.
 51. Thenon-human animal of claim 43, which is a mouse.
 52. The non-human animalof claim 43, which is an ungulata.
 53. The non-human animal of claim 43,which is a bovine.
 54. The non-human animal of claim 43, which is anovine